Name: a guide to modern quantitative fluorescent western blotting with troubleshooting strategies
Uploaded: 2014-11-20T16:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

<p class="jove_content"> Nous tenons à remercier pour le soutien financier qui suit: BBSRC Institut stratégique de financement des programmes - CF &amp; TMW; Financement BBSRC Est Bio DTP - LG; Le Darwin Trust of Edinburgh - MLH. Nous tenons également à remercier le Dr Barry McColl pour permission d&#39;inclure l&#39;optimisation TREM2 dans ce manuscrit. </p>

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> <em style='font-style: italic'>Biotechnology</em>. <b>1992</b> (24), 145-149 (1979).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Protein Protocols on CD-ROM</em>. , (1998).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> <em style='font-style: italic'>Curr Protoc Mol Biol. Chapter</em>. <b>10</b>, 10 (2008).</li><li>Eaton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+protein+analysis+as+a+reliable+loading+control+for+quantitative+fluorescent+Western+blotting.">Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.</a> <em style='font-style: italic'>PLoS One</em>. <b>8</b> (8), 72457 (2013).</li><li>Suzuki, O., Koura, M., Noguchi, Y., Uchio-Yamada, K., Matsuda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sample+mixtures+for+standard+curve+creation+in+quantitative+Western+blots.">Use of sample mixtures for standard curve creation in quantitative Western blots.</a> <em style='font-style: italic'>Exp. Anim</em>. <b>60</b>, 193-196 (2011).</li><li>Colella, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Stain-Free+gels+with+traditional+immunoblot+loading+control+methodology.">Comparison of Stain-Free gels with traditional immunoblot loading control methodology.</a> <em style='font-style: italic'>Anal Biochem</em>. <b>430</b> (2), 108-110 (2012).</li><li>Zellner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+Western+blotting+for+quantitation+of+protein+biomarkers+in+clinical+samples.">Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.</a> <em style='font-style: italic'>Electrophoresis</em>. <b>29</b>, 3621-3627 (2008).</li><li>Kielar, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+correlates+of+axonal+and+synaptic+pathology+in+mouse+models+of+Batten+disease.">Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.</a> <em style='font-style: italic'>Hum Mol Genet</em>. <b>18</b>, 4066-4080 (2009).</li><li>Mutsaers, C. A., Lamont, D. J., Hunter, G., Wishart, T. M., Gillingwater, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+proteomics+identifies+Calreticulin+and+GRP75/Mortalin+as+peripherally+accessible+protein+biomarkers+for+spinal+muscular+atrophy.">Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.</a> <em style='font-style: italic'>Genome Med</em>. <b>5</b> (10), 95 (2013).</li><li>Wishart, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+ubiquitin+homeostasis+and+&#946;-catenin+signaling+promote+spinal+muscular+atrophy.">Dysregulation of ubiquitin homeostasis and &#946;-catenin signaling promote spinal muscular atrophy.</a> <em style='font-style: italic'>J Clin Invest</em>. <b>124</b> (4), 1821-1834 .</li><li>Mathews, S. T., Plaisance, E. P., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+systems+for+westerns:+chemiluminescence+vs.+infrared+detection.">Imaging systems for westerns: chemiluminescence vs. infrared detection.</a> <em style='font-style: italic'>Methods Mol Biol</em>. <b>536</b>, 499-513 (2009).</li><li>Hawes, J. J., Brunzell, D. H., Wynick, D., Zachariou, V., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GalR1,+but+not+GalR2+or+GalR3,+levels+are+regulated+by+galanin+signalling+in+the+locus+coeruleus+through+a+cyclic+AMP-dependent+mechanism.">GalR1, but not GalR2 or GalR3, levels are regulated by galanin signalling in the locus coeruleus through a cyclic AMP-dependent mechanism.</a> <em style='font-style: italic'>J Neurochem</em>. <b>93</b> (5), 1168-1176 (2006).</li><li>Bond, D., Primrose, D. A., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+signaling+events+in+Drosophila+S2+cells.">Quantitative evaluation of signaling events in Drosophila S2 cells.</a> <em style='font-style: italic'>Biol Proceed Online</em>. <b>10</b> (1), 20-28 (2008).</li><li>Wishart, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+proteomics+analysis+of+synaptic+proteins+identifies+potential+cellular+targets+and+protein+mediators+of+synaptic+neuroprotection+conferred+by+the+slow+Wallerian+degeneration+(Wlds)+gene.">Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.</a> <em style='font-style: italic'>Mol Cell Proteomics</em>. <b>6</b>, 1318-1330 (2007).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+western+in+town:+a+contemporary+twist+on+the+classic+western+blot+analysis.">The fastest western in town: a contemporary twist on the classic western blot analysis.</a> <em style='font-style: italic'>J. Vis. Exp</em>.  (84), 51149 (2014).</li></ol>

<p class='jove_content'>The authors have nothing to disclose.</p>

<p class="jove_content"> Une attention particulière et de la planification est essentielle avant toute expérience et peuvent en fin de compte déterminer le succès de la technique utilisée. Les progrès dans la détection de la protéine à l&#39;aide de la Banque mondiale peut présenter une pléthore d&#39;obstacles potentiels en essayant de choisir les anticorps, de transfert et de visualisation des méthodes appropriées à utiliser. Heureusement, en utilisant une liste de contrôle attentif et des mesures de contrôle appropriées QFWB peut être utilisée en routine pour déterminer la présence de la protéine et des différences d&#39;expression de plus en plus subtiles entre les échantillons. Ce protocole fournit un guide complet fluorescent western blot quantitatif ainsi que quelques stratégies de dépannage pour éviter et / ou de surmonter quelques-uns des nombreux écueils les plus courants associés. </p><p class="jove_content"> Les étapes essentielles utilisées pour maintenir la sensibilité et obtenir vraiment des mesures quantifiables et comparables comprennent: 1) l&#39;extraction des protéines robuste à partir d&#39;échantillons de tissus; 2) la préparation des échantillons; 3) précise loadin de protéinesg déterminée par l&#39;analyse des protéines totales; 4) un transfert optimal de protéines en utilisant I-Blot; 5) la préparation d&#39;anticorps primaires et secondaires dans le tampon de blocage contenant 0,1% de Tween 20, et 6) visualisation correcte et analyse à l&#39;aide d&#39;un imageur infrarouge et les logiciels associés. </p><p class="jove_content"> Détection fluorescente infrarouge est vraiment quantitative et offre une plus grande sensibilité par rapport à d&#39;autres techniques de détection ECL traditionnelles <sup>3,11.</sup> Ce système de détection est à plusieurs facettes, et en tant que tel ne se limite pas à QFWB. Ce système est capable de formation d&#39;image de marquage immunohistochimique à faible puissance permettant la visualisation et la quantification des coupes de tissus entiers <sup>12.</sup> Ceci est une zone de développement potentiel futur en termes de résolution qui pourrait voir l&#39;imagerie rouge lointain rivalisant avec capture d&#39;immunofluorescence conventionnelle avec des microscopes conventionnels en termes d&#39;évaluation quantitative. </p><p class="jove_content"> Cependant, avec une plus grande sensibilité à des changements subtils dans protein expression, il est essentiel de veiller à la variabilité est réduite au minimum et les mesures de contrôle sont strictes avec des protocoles robustes. Cela commence avec l&#39;extraction des protéines rigoureuse de l&#39;échantillon de tissu suivie par la production de protéines colorées au total gels à fournir l&#39;assurance que le chargement des échantillons est uniforme, optimisation des anticorps primaires et secondaires afin de déterminer si la détection est réel et tester les directives du fabricant en ce qui concerne le transfert fois pour obtenir le transfert de la protéine efficace. </p><p class="jove_content"> Néanmoins, même lorsque les conditions de la Banque mondiale sont optimisés, il peut y avoir encore des problèmes associés à la gestion des westerns qui peuvent ne pas avoir été pleinement explorées ici. Ceux-ci comprennent, sans s&#39;y limiter, les facteurs, y compris la solubilisation de la protéine et le choix de tampon d&#39;extraction. Des tampons peuvent interférer avec des dosages de la concentration de protéine, et certains tissus sont particulièrement difficiles à solubiliser, ce qui nécessite des techniques plus robustes telles que l&#39;utilisation de macération automatiséTing emballage étanche, tel que M TUBES ensemble avec un dissociateur Mac. En outre, des mesures de contrôle simples pour le stockage de la matière extraite et non extraite à -80 ° C peuvent faire la différence entre l&#39;obtention d&#39;étiquetage optimale immédiatement après l&#39;extraction et ayant pauvres résultats semaines plus tard. </p><p class="jove_content"> Méthodes de QFWB modernes se sont révélés être plus sensible pour capturer des différences subtiles dans l&#39;expression des protéines et permettent aux plus polyvalent simultanée double étiquetage <sup>3</sup> par rapport aux techniques plus anciennes comme ECL. Il est essentiel que les protocoles de transfert de Western sont robustes et facilement reproductible pour la quantification précise et l&#39;analyse statistique. Ce protocole est sensible et suffisamment robuste pour être utilisée en routine pour la détection de protéines à travers une série d&#39;échantillons de tissus et d&#39;espèces différentes et <sup>3</sup> permet la quantification des protéines de faible abondance et élevés à l&#39;intérieur de la même QFWB réduisant par conséquent l&#39;utilisation des consommables ainsi que le temps pour l&#39;expérience <sup>1</sup>. 1 En outre, la sensibilité accrue de cette technique permet la validation de plus en plus populaire - études <em>omiques</em> <sup>9,14</sup> Cependant, l&#39;exactitude est cruciale et l&#39;inclusion de mesures de contrôle appropriées doivent être respectées évitant ainsi l&#39;acquisition de données erronées. </p>

<p class="jove_content"> Western Blot (WB) est une technique d&#39;analyse développé à l&#39;origine dans les années 1970 pour déterminer la présence ou l&#39;absence d&#39;une protéine d&#39;intérêt dans un échantillon biologique complexe, comme un homogénat de tissu <sup>1.</sup> Communément dénommé immunoblot des protéines, en raison de la clé interaction anticorps-antigène, la méthode consiste en cinq étapes distinctes: 1) la séparation électrophorétique des protéines par leur point isoélectrique; 2) le transfert à une nitrocellulose ou polyfluorure de vinylidène (PVDF); 3) le marquage en utilisant un anticorps primaire spécifique à la protéine d&#39;intérêt; 4) incubation avec un anticorps secondaire dirigé contre l&#39;anticorps primaire; et 5) la visualisation. </p><p class="jove_content"> méthodologie de visualisation a évolué avec le temps pour améliorer la sécurité et de la sensibilité. Certains des premiers Western Blots ont été réalisées en utilisant la radio balises marquées qui ensuite évolué vers les chemilluminescent plus largement utilisé (ECL) des méthodes colorimétriques et puis. Radioactivity a été marqué directement sur ​​les sondes pour des antigènes spécifiques alors que les méthodes colorimétriques et ECL utilisent une technique de marquage indirect avec un rapporteur enzymatique tel que la peroxydase de phosphatase alcaline ou de la streptavidine de raifort (HRP) <sup>2.</sup> L&#39;intensité de l&#39;étiquetage du produit luminescent ou chromogène est mesurée en utilisant la densitométrie par lequel la force du signal, fort ou faible, plus ou moins indique la présence de la protéine d&#39;intérêt dans l&#39;échantillon. ECL est la méthode plus sensible et donc favorisée mais toutes les <sup>deux</sup> trois méthodes ont été initialement mis au point sur ​​le film à rayons X avec des techniques d&#39;imagerie numérique plus sophistiqué ensuite établies <sup>3.</sup> L&#39;avancement de l&#39;imagerie numérique de WB a non seulement permis à un chercheur de déterminer la présence ou l&#39;absence de leur protéine d&#39;intérêt, mais a également permis une déduction au niveau de l&#39;expression d&#39;une protéine choisie lorsqu&#39;on les compare aux autres échantillons et peut donc être considéré comme «semi-quantitative &amp; #8221 ;. Récemment, une technique de transfert de Western véritablement quantitative et plus sensibles ont été mis au point de sorte que le niveau de fluorescence mesurée est directement liée à la quantité et à l&#39;expression d&#39;une protéine unique dans un échantillon: quantitatif fluorescent western blot (QFWB). </p><p class="jove_content"> Lorsque l&#39;on compare avec l&#39;étiquetage QFWB ECL, l&#39;utilisation d&#39;un anticorps secondaire fluorescent génère un profil de détection linéaire <sup>4.</sup> Ceci est en contraste avec les techniques ECL où la linéarité du signal se produit généralement avec des charges faibles en protéines est inférieure à 5 ug et la saturation de signal directement liés à l&#39;expression des protéines, <em>à savoir,</em> avec des gènes domestiques exprimés de manière ubiquitaire <sup>5,6.</sup> Cette disparité est très probablement dû à un plus grand nombre de sites de liaison disponibles pour un substrat ECL avidine à se lier à un secondaire biotinylé, ce qui entraîne un risque plus élevé de potentiel de saturation du signal. Ceci est l&#39;une des principales raisons de la base ECL immuno étant désignées comme étant ONLy &quot;semi-quantitative» <sup>7.</sup> Le point de signal de saturation est d&#39;une importance critique pour la mesure des différences subtiles dans les niveaux d&#39;expression et peut conduire à des mesures inexactes. Au cours des dernières années, l&#39;avènement des techniques protéomiques répandues détaillant de plus en plus la sensibilité et l&#39;identification des différences d&#39;expression subtiles a entraîné une dépendance croissante sur vraiment quantitative Western blot pour des expériences de validation <sup>8,9.</sup> L&#39;application de la méthodologie sensible, dynamique et véritablement quantitative est donc crucial. </p><p class="jove_content"> Beaucoup de &quot;optimisés&quot; méthodes de transfert de Western ont été utilisés par des laboratoires indépendants, qui se révèlent souvent difficiles à mettre en place ou de reproduire en raison d&#39;ajustements méthodologiques subtils qui peuvent ne pas être dans les protocoles officiels et documentés. Ceci est un protocole établi et robuste pour QFWB et fournit des stratégies utiles pour la résolution des problèmes communs tha plust peut survenir au cours de la mise en œuvre. </p><p class="jove_content"> Ce protocole a été optimisé pour une utilisation avec des homogénats de cerveau de souris, mais a depuis été utilisé efficacement dans un large éventail d&#39;échantillons de tissus et les espèces <sup>4,9,10.</sup> Variations de protocole de potentiel nécessaires pour résoudre les problèmes spécifiques sont inclus. </p>

<table><tbody><tr><td>RIPA Buffer</td><td>Fisher Scientific UK Ltd</td><td>10230544</td><td></td></tr><tr><td>M Tubes</td><td>Miltenyi Biotec Inc.</td><td>130-093-236</td><td></td></tr><tr><td>iBlot Transfer Stack, PVDF Regular</td><td>Life Technologies, UK</td><td>IB401001</td><td></td></tr><tr><td>MagicMark XP Western Protein Standard (20-220 kDa)</td><td>Life Technologies, UK</td><td>LC5602</td><td>Use in gel 1 for Western blotting</td></tr><tr><td>SeeBlue Pre-stained protein standard</td><td>Life Technologies, UK</td><td>LC5625&amp;nbsp;</td><td>Use in gel 2 Total protein stained gel</td></tr><tr><td>NuPAge LDS Sample buffer 4x</td><td>Life Technologies, UK</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20x)</td><td>Life Technologies, UK</td><td>NP0002</td><td></td></tr><tr><td>NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well</td><td>Life Technologies, UK</td><td>NP0322BOX</td><td></td></tr><tr><td>Phosphate buffered saline tablet</td><td>Sigma-Aldrich, UK</td><td>P4417-100TAB</td><td></td></tr><tr><td>Micro BCA, Protein Assay Kit</td><td>Fisher Scientific UK Ltd</td><td>10249133</td><td></td></tr><tr><td>Odyssey blocking buffer</td><td>Li-Cor Biosciences</td><td>P/N 927-40000</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg</td><td>Li-Cor Biosciences</td><td>926-68071</td><td></td></tr><tr><td>IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg&amp;nbsp;</td><td>Li-Cor Biosciences</td><td>926-68072</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5 mg</td><td>Li-Cor Biosciences</td><td>926-32211</td><td></td></tr><tr><td>IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5 mg</td><td>Li-Cor Biosciences</td><td>926-32214</td><td></td></tr><tr><td>ODYSSEY CL Infra-red imager</td><td>Li-Cor Biosciences</td><td></td><td>Call for quotation</td></tr><tr><td>iBlot 7-Minute Blotting System</td><td>Life technologies, UK</td><td></td><td>This model is no longer in production</td></tr><tr><td>InstantBlue Protein stain</td><td>Expedeon, UK</td><td>ISB1L</td><td></td></tr><tr><td>Revitablot western blot stripping buffer&amp;nbsp;</td><td>Rockland Immunochemicals Inc.</td><td>MB-085-0050</td><td></td></tr></tbody></table>

a guide to modern quantitative fluorescent western blotting with troubleshooting strategies

<p class="jove_content"> La fin des années 1970 ont vu la première utilisation déclarée publiquement du western blot, une technique pour évaluer la présence et l&#39;abondance relative des protéines spécifiques dans des échantillons biologiques complexes. Depuis lors, la méthodologie western blot est devenue un élément commun de la biologie moléculaire répertoire expérimental. Une recherche rapide de PubMed en utilisant le terme &quot;western blot&quot; suggère que plus de 220 000 manuscrits publiés ont fait usage de cette technique d&#39;ici l&#39;an 2014. Surtout, les dix dernières années ont vu des avancées d&#39;imagerie techniques couplées avec le développement marqueurs fluorescents de sensibles qui ont permis d&#39;améliorer la sensibilité et a donné encore plus de plages de détection linéaire. Le résultat est une fluorescence maintenant vraiment quantifiable fondé Western Blot (QFWB) qui permet aux biologistes d&#39;effectuer une analyse comparative de l&#39;expression avec une plus grande sensibilité et de précision que jamais auparavant. Beaucoup de &quot;optimisé&quot; western blotméthodes existent et sont utilisées dans différents laboratoires. Ceux-ci se révèlent souvent difficiles à mettre en œuvre en raison de l&#39;exigence de modifications procédurales subtiles mais sans papiers. Ce protocole fournit une description complète de la méthode d&#39;QFWB établi et robuste, avec des stratégies de résolution des problèmes. </p>

<p class="jove_content"> Comme la sensibilité à QFWB et la plage linéaire de détection est supérieure à la détection ECL classique, il ya un certain nombre de mesures de contrôle qui sont essentiels pour assurer l&#39;exactitude des données sont collectées, facilitant ainsi l&#39;interprétation efficace. Tout d&#39;abord, l&#39;inclusion des échantillons témoins positifs comme représenté sur <strong>la figure 1.</strong> D&#39;autre part, l&#39;optimisation du transfert de garantir un mouvement équivalent de protéines de haut et de bas poids moléculaire à partir du gel à la membrane comme présenté dans <strong>la figure 2.</strong> En troisième lieu, l&#39;optimisation d&#39;anticorps, en particulier secondaire des anticorps dont l&#39;optimisation est souvent négligé, mais qui peuvent produire non spécifique bandes capables d&#39;interférer avec l&#39;interprétation correcte de la protéine (s) d&#39;intérêt. Voir <strong>Figure 3.</strong> Quatrièmement, il peut aussi être le cas que quand une protéine apparaît indétectable mais devrait être présente, cela peut aussi être une question d&#39;anticorps secondaire qui peut être corrigé en utilisant simplement un raïs secondairesed dans un hôte de différentes espèces. Voir <strong>figure 4.</strong> Cinquièmement, l&#39;étiquetage de la protéine totale et l&#39;analyse est une méthode beaucoup plus robuste et quantifiable par rapport à l&#39;utilisation unique de protéine traditionnel (s) qui sont exprimés de façon ubiquitaire à des étalons de référence internes <sup>3.</sup> Beaucoup de ces protéines simples ont été trouvés à être exprimés de manière différentielle dans des modèles de maladies neurodégénératives, ainsi qu&#39;entre les échantillons de tissus différents et l&#39;uniformité d&#39;expression peut modifier dans le même tissu <sup>3.</sup> Par conséquent, la production d&#39;un gel de commande de charge confirme l&#39;uniformité de la charge de l&#39;échantillon lorsqu&#39;il est combiné avec une analyse des protéines totales par la comparaison et la quantification de la charge de protéine dans chaque ligne à différentes gammes de poids moléculaires mesurés par rapport à chaque échantillon pour indiquer l&#39;erreur standard comme le montre <strong>la figure 6</strong> . Fait important, l&#39;ensemble de ces techniques et des contrôles de dépannage ne sont efficaces que la sensibilité et la cohérence de l&#39;analyse tutils appliquées par l&#39;opérateur <strong>(figure 5).</strong> Enfin cette technique se prête bien à l&#39;arrachement et re-sondage de membranes avec plus de souplesse que ECL en raison de facteurs y compris, mais sans s&#39;y limiter, une sensibilité accrue, de fond réduit, la détection de double couleur et la stabilité de la membrane dans des conditions de stockage à long terme. Voir <strong>la Figure 7.</strong> </p>

Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

<p class='jove_content'>The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.</p>

Un Guide to Modern quantitative fluorescente Western blot avec des stratégies de dépannage

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

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Research

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Biology

51.0K vues.  University of Edinburgh. The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Vidéo: A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Lab Techniques

<h4>Measurement Accuracy</h4>
<p>The ability to repeat an experiment and get the same results, or <em>reproducibility</em>, is essential in scientific research. However, it&#8217;s impossible to repeat an experiment if you don&#8217;t know how you did it. Thus, scientists keep detailed records of their experiments in lab notebooks. These records include important information like the amount of each material used and descriptions of each step of the procedure.</p>
<p>Equipment, like balances and volumetric or graduated glassware, is used to measure solid and liquid compounds for experiments. The precision of each measurement is limited by the equipment used. Balances and other equipment with digital readouts automatically display values at the maximum precision that they can support. The precision of graduated glassware and rulers depends on the increment, which is the amount represented by the smallest distance between two marks.</p>
<p>The surface of most liquids curves upwards at the edges in glass containers. This type of curved liquid surface is called a concave meniscus. When the edges of the liquid curve down, it is called a convex meniscus. When measuring liquid volumes, the reference point for the top of the liquid is the bottom of a concave meniscus or the top of a convex meniscus. To accurately identify the position of the meniscus, liquid volumes are read by looking at the liquid&#8217;s surface from the side at eye-level. Looking at the liquid from above or below will cause the liquid surface level to appear higher or lower than it actually is.</p>
<p>When the reference point of the meniscus touches a mark on graduated or volumetric glassware, the glassware is holding the volume defined for that mark. Volumetric glassware is designed to measure a specific volume, so it only has one mark. Graduated glassware is designed to measure a range of volumes, so it has many marks, or <em>graduations</em>, which are labeled at regular intervals. Both graduated and volumetric glassware may have additional manufacturer-provided information about their precision.</p>
<h4>Calibration and Uncertainty</h4>
<p>Manufacturers calibrate equipment to ensure that the measurements are accurate to within a specified range of uncertainty. For example, a high-precision analytical balance may read out to four decimal places (0.0000 g) with an uncertainty of &#177; 0.0001 g. A reading of 0.0345 g indicates that the true value is between 0.0344 g and 0.0346 g.</p>
<p>Glassware typically has its measurement uncertainty printed on it. To accurately record volume measurements, you must consider both the increment and the uncertainty. For example, a precise volume measurement in a 100 mL graduated cylinder with 1 mL increments and an uncertainty of &#177; 0.5 mL would be recorded to the tenths place (000.0 mL &#177; 0.5 mL). If the reference point of the meniscus falls between two marks, you should estimate a value for the tenths place; otherwise, leave it as 0. Since temperature affects volume, the temperature for which the glassware is calibrated will be printed on it.</p>
<p>Glassware is calibrated either <em>to contain</em> (TC) or <em>to deliver</em> (TD). TC glassware holds the specified volume of liquid when it is filled to the mark, but a small amount of liquid will be left behind when it is poured into another container. Solutions are often prepared in TC glassware because the accuracy of the solution volume affects the accuracy of the concentration. Volumetric flasks are typically calibrated to contain.</p>
<p>TD glassware holds a little more than the amount of liquid specified when it is filled to the mark, but it dispenses only the specified volume. Thus, TD glassware should not be emptied completely when dispensing liquids. This type of glassware is useful for transferring a precise volume of liquid to another container. Both graduated and volumetric pipettes are typically calibrated to deliver.</p>
<p>Unless otherwise specified, the uncertainty of a number is assumed to be on the scale of the last digit. Thus, it&#8217;s particularly important to report the uncertainty of a measurement if the uncertainty is on a larger scale. For rulers and graduated glassware without a known uncertainty, the uncertainty is estimated as half of the smallest increment. The uncertain digit is based on where the meniscus falls between two tick marks.</p>
<h4>Significant Figures</h4>
<p>Significant figures are the numbers in a value that are <em>meaningful</em>, or essential for expressing that value with the appropriate precision. All numbers other than leading zeroes (<strong>0.00</strong>1), trailing zeroes (1,<strong>000</strong>), or scientific notation multipliers (<strong>10</strong><sup>x</sup>) are always significant.</p>
<p>Leading zeroes are never significant because they can be removed by rewriting the number either in scientific notation or, if it is an SI unit, in smaller units. For example, there are only three significant figures in the value 0.00123 m because it can be rewritten as either 1.23 &#215; 10<sup>-3</sup> m or 1.23 mm without losing any information. Note that the scaling factor between different multiples of the same SI unit does <em>not</em> affect the number of significant figures.</p>
<p>Trailing zeroes are significant when they are before or after a decimal point. For example, the measurement 100.110 mL has six significant figures and 100.0 mL has four. Trailing zeroes in a number written without a decimal point are assumed to be insignificant unless they are marked otherwise. There are several different conventions for marking significant zeroes in those cases, but to avoid ambiguity it is advisable to rewrite those values in scientific notation. If the measurement is in SI units, use larger units.</p>
<p>The precision of a measurement limits the precision of any values calculated from it. A calculated value has the same number of significant figures as the <em>least</em> precise measurement or value used in its calculation. If the result has too many significant figures, it is rounded to the appropriate precision. If the result has too few significant figures, it is extended with significant trailing zeroes to preserve the precision. The significant figures of experimentally derived values, like molar mass, density, or some unit conversion factors, must also be considered when they are used in calculations.</p>
<p>The number of significant figures in a calculated value is <em>not</em> limited by physical or mathematical constants in formulas or exact numbers, like the number of data points acquired in an experiment. For example, the formula for calculating the volume of a sphere is V = 4/3&#960;r<sup>3</sup>. The number of significant figures in the calculated volume is <em>only</em> affected by the number of significant figures in the radius measurement <strong>r</strong>. 4/3&#8201; and &#960; are constants, and <sup>3</sup> is simply notation for a mathematical operation.</p>
<p>Defined conversion factors are also treated as constants. For instance, the inch is defined as exactly 25.4 millimeters; therefore, the number of significant figures of the converted value depends only on the original measurement. Always check whether the conversion factor is exactly defined or an experimental value when converting between units.</p>
<p>For simple addition and subtraction of measurements, we use significance arithmetic to determine the number of significant figures. In this case, the answer has as many decimal places as the measurement with the fewest decimal places <em>regardless of the number of significant figures</em>. Thus, 15,643.7 mL + 0.613 mL = 15,644.3 mL.</p>
<p>For multi-step calculations or complex equations that you break into smaller parts to solve, you should keep at least one or two insignificant figures on the intermediate values. For example, if you calculate the values of the numerator and denominator of a fraction separately before dividing them, you should retain the insignificant figures of the numerator and denominator when you divide them. This minimizes <em>rounding error</em>, which is the difference from the number you would get if you entered the entire formula into your calculator at once. If you write down these intermediate values, you should note which digits are insignificant.</p>
<h4>References</h4>
<p>Harris, D.C. (2015). <em>Quantitative Chemical Analysis</em>. New York, NY: W.H. Freeman and Company.</p>

Accuracy, Precision, Uncertainty, and Significant Figures - Concept

Techniques de laboratoire

Measurement Accuracy
The ability to repeat an experiment and get the same results, or reproducibility, is essential in scientific research. However, ...

Enseignement

JoVE Lab Manual

Chimie

Thin-Layer Chromatography

<p><strong>Chromatography</strong></p>
<p>Chromatography is a technique used in organic chemistry to separate compounds in a mixture based on their differences in solubility between two different phases. The concept is similar to liquid-liquid extraction, except in chromatography, the two phases consist of the stationary phase and the mobile phase. The stationary phase is a solid &#8212; typically a microscale hydrogel bead &#8212; while the mobile phase is a carrier solvent.</p>
<p>In traditional chromatography, the stationary phase is packed into a vertical column, and the solution mixture introduced at the top of the column. As the mixture flows through the stationary phase, the compounds partition between the stationary phase and the mobile phase based on their structures and polarities, forming discrete bands. Solutes that interact weakly with the stationary phase move more quickly through the column and exit &#8212; or elute &#8212; first. Solutes that interact more strongly with the stationary phase move slowly through the column and elute later. The bands can be collected individually in order to isolate and purify compounds in a mixture.</p>
<p>There are several types of chromatography, each harnessing a different chemical property to achieve separation. For example, in ion-exchange chromatography, the stationary phase beads can be positively or negatively charged, attracting molecules with opposite charge only. In size exclusion chromatography, the porous stationary phase is composed of beads such as agarose or dextran polymers. Smaller molecules can enter the pores more readily, while the larger molecules flow past the pores and elute faster.</p>
<h4><strong>Thin-Layer Chromatography</strong></h4>
<p>Thin-layer chromatography (TLC) is a type of chromatography technique that separates compounds based on their polarity. Like traditional chromatography, there are three components of a TLC system: the stationary phase, the mobile phase, and the solute. However, unlike traditional chromatography, the stationary phase is arranged in a thin layer on a plate rather than packed into a column. TLC most often uses polar silica gel, a form of silicon dioxide, as the stationary phase. The stationary phase forms hydrogen bonds due to the OH groups on its surface.</p>
<p>First, a starting line is drawn on the bottom of the TLC plate using a pencil. The compounds or mixture being analyzed are spotted on the starting line using a thin capillary. Then, the bottom of the plate is immersed in the mobile phase, which is usually an organic solvent that is less polar than the stationary phase. The solvent travels up the plate by capillary action, passing the solute spots and carrying some of each component with it.</p>
<p>As the solvent travels up the plate, the components partition to either the mobile phase or the stationary phase. If the component is polar, it interacts more with the polar stationary phase. It travels slowly and only moves a short distance on the TLC plate. If the component of the sample is less polar &#8212; and more soluble in the mobile phase than it is in the stationary phase &#8212; it interacts more with the mobile phase and travels farther on the TLC plate. The extent of the polarity of the component and the mobile phase are essential to understanding and predicting the separation.</p>
<p>TLC plates typically contain a UV-reactive fluorescent dye that will glow under a UV source of 254 nanometers. Therefore, TLC plates can be analyzed by observing them under UV light. Compounds within the TLC plate, such as the solutes of interest, will show up as dark spots compared to a green background. By circling the spots with a graphite pencil, the distance the compounds traveled relative to the solvent front can be measured. The spot of the organic compound, if not fluorescent itself, masks the fluorescence of the plate and shows up as a dark spot. Some organic compounds are UV-active and emit light when exposed to UV light. These are typically conjugated compounds, meaning those with alternating double and single bonds, and can be identified by the wavelength emitted.</p>
<h4><strong>Retardation Factor</strong></h4>
<p>By analyzing the retardation factor (R<sub>f</sub>) of a component with a specific solvent, an unknown solute can be determined using TLC. The retardation factor is the ratio of the distance traveled by a component to the distance traveled by the mobile phase.</p>
<p style="text-align: center;"><img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/62/62_Concepts_1.jpg" /></p>
<p>The distance traveled by the solute is measured from the starting line to the center point of the spot, and the distance traveled by the mobile phase is measured from the same starting line to the solvent front. The retardation factor of a compound is dependent on the mobile phase used. The retardation factor is large for compounds that are highly non-polar with a non-polar mobile phase. Low retardation factor values are seen for polar components with a non-polar mobile phase.</p>
<p>In the case of a highly non-polar mobile phase, some polar components may not move at all. This results in an extremely low retardation factor and an insufficient separation. A highly polar mobile phase causes the compound to move with the solvent and yields an extremely high retardation factor. This results in very little separation between the components.</p>
<p>For separation to be effective, the retardation factors of the components should be about 0.3 - 0.7 apart. To find the efficient mobile phase, trial and error is employed. Often, a mixture of two solvents proves most effective.</p>
<h4><strong>References</strong></h4>
<ol>
	<li style="font-size:17px">Harris, D.C. (2015). <em>Quantitative Chemical Analysis.</em> New York, NY: W.H. Freeman and Company.</li>
</ol>

Thin-Layer Chromatography: Experimental Setup and Separation - Concept

Chromatographie sur couche mince

Chromatography
Chromatography is a technique used in organic chemistry to separate compounds in a mixture based on their differences in solubility between ...

Column Chromatography

<p><strong>Column Chromatography</strong></p>
<p>Multiple techniques exist to purify and separate compounds in an organic chemistry laboratory. One of the most reliable separation techniques for microscale separations, where less than 10 grams of the compound is available for analysis, is column chromatography. This technique separates compounds in a mixture based on their physical properties, such as their solubility, polarity, hydrophobicity, size, and charge.</p>
<h4><strong>Chromatography Basics</strong></h4>
<p>The main components of any chromatography system are the stationary phase, the mobile phase, and the sample mixture to be analyzed. In column chromatography, the stationary phase is typically microscale beads, which are packed uniformly in a vertical column. A continuous flow of the mobile phase, also known as the solvent, is added to the top of the column, which flows through the stationary phase via gravity or at a controlled flow rate by a pump.</p>
<p>The sample is dissolved in a small amount of the solvent and then added to the top of the column. More solvent is added to force the sample to flow through the stationary phase. The compounds in the mixture partition between the mobile phase and the stationary phase based on their differing properties and form discrete bands.</p>
<p>Compounds with strong interactions with the stationary phase will move slower than compounds with weak interactions with the stationary phase. Thus, compounds with weak interactions will exit the column, or elute, earlier than those with strong interactions. The strength of a compound&#8217;s interaction with the stationary phase is defined by the retardation factor (R<sub>f</sub>), which is the ratio of the distance traveled by a component to that traveled by the mobile phase. The retardation factor is determined by first performing thin layer chromatography.</p>
<p style="text-align: center;"><img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/63/63_Concepts_1.jpg" /></p>
<p>A high retardation factor value indicates that the sample interacts strongly with the stationary phase and takes a long time to elute. A low retardation factor value indicates that the sample does not interact with the stationary phase very strongly and elutes more quickly. Since compounds partition into individual bands and elute from the column at different times due to their different R<sub>f</sub> values, they can be isolated individually by collecting the solvent from under the column in fractions.</p>
<h4><strong>Column Chromatography Considerations</strong></h4>
<p>A chromatography column is a vertically oriented glass or plastic tube. The size of the column can range from a few centimeters (such as a Pasteur pipette) to meters (such as with industrial columns). The diameter of the column depends on the amount of sample to be separated, whereas the length of the column depends on the difficulty of separation. The most efficient partitioning into discrete bands requires thin sample layers; therefore, determining the appropriate diameter is a vital step. Loading too much sample volume relative to the column&#8217;s diameter will create broader and less defined bands than the same volume of sample in a column with a larger diameter.</p>
<p>When choosing the length of the column, the R<sub>f</sub> values of the components must be considered. Components with similar retardation factors may be challenging to separate and require a longer column to resolve the individual bands. Compounds with very different retardation factors may not need a long column, as they should partition easily. When selecting the stationary phase, it is important to choose one that results in different retardation factors for each component.</p>
<p>Preparing the column is the most critical part of running a chromatography column. The mass of the stationary phase packed in the column should be at least 20x that of the sample. If the column does not have a porous disk at the bottom, it should be packed with cotton and a thin layer of sand. This prevents the loss of the stationary phase through the exit of the column.</p>
<p>There are two methods to pack the column: the dry packing method and the slurry method. In the dry method, the stationary phase is transferred into the column in its powder form. The column is then washed several times with the mobile phase/solvent to ensure that the solvent penetrates every part of the silica gel column. This method, if not done correctly, can increase the probability of dry patches, channels, and air bubbles forming in the column. These defects will affect the ability of the column to partition components of a mixture.</p>
<p>The slurry method provides a more uniform packed column that does not contain air bubbles, dry areas, and channels. For this method, the stationary phase is mixed with a solvent to form a consistent slurry. The slurry is then transferred to the column while tapping at the sides to get rid of any air bubbles formed. The stopcock of the column, if there is one, should be open during this step to allow the solvent to drain.</p>
<h4><strong>Silica Gel Chromatography</strong></h4>
<p>Many different properties are exploited to separate a mixture of compounds, such as size, charge, and hydrophobicity. One property used to separate compounds in organic chemistry is polarity. For this, silica gel and alumina gel are the most commonly used stationary phases. Silica gel is extremely polar and forms strong dipole-dipole interactions with polar compounds. In addition, silica gel can form hydrogen bonds with the analyte due to the presence of -OH groups on its surface.</p>
<p>Components of the mixture that are most polar bind strongly to the silica gel and travel slowly through the column, while non-polar components are more soluble in the mobile phase and travel quickly through the column. Thus, it is essential that the components of the mixture have different polarities. Components that interact strongly with the stationary phase are eluted by flowing a polar mobile phase through the column.</p>
<h4><strong>References</strong></h4>
<ol>
	<li style="font-size:17px">Shuler, M.L., Kargi, K., DeLisa, M. (2017). <em>Bioprocess Engineering, Basic Concepts.</em> Upper Saddle River, NJ: Prentice Hall.</li>
	<li style="font-size:17px">Harris, D.C. (2015).<em> Quantitative Chemical Analysis.</em> New York, NY:<em> W.H. Freeman and Company.</em></li>
</ol>

Column Chromatography: Experimental Setup and Separation - Concept

Chromatographie en colonne

Column Chromatography
Multiple techniques exist to purify and separate compounds in an organic chemistry laboratory. One of the most reliable separation ...

Synthesis of Luminol

<p>Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. The difference in energy between reactants and products is known as the enthalpy of the reaction. This is found by calculating the difference in energies between the products and the reactants, or &#916;H. If &#916;H is positive, the reaction is endothermic; if it is negative, the reaction is exothermic. While chemical reactions are traditionally thought of as a conversion of reactants into products, many reactions take place in multiple steps, forming intermediates. In chemiluminescent reactions, these intermediates transition from a high energy state to the ground state, releasing a photon of light that may be visible as the reaction progresses.</p>
<h4><strong>Atomic Energy Levels</strong></h4>
<p>Niels Bohr, a Danish physicist, proposed the theory that an electron orbiting the nucleus of the atom can occupy only certain orbits or energy levels. An atom with all of its electrons in the lowest possible energy level is said to be at its ground state. When an electron occupies an energy level higher than the ground state, the atom is in its excited state.</p>
<p>A key assumption in Bohr&#8217;s theory is that an electron remains in the ground state until it is disturbed. Thus, an electron is only elevated to an excited state by absorbing energy. When the electron relaxes back down to the ground state, it releases that energy. Often, this is in the form of a photon of light, whose wavelength is directly related to the energy difference between the excited and ground states.</p>
<p>When an electron is excited to a higher energy level by absorbing light of a certain energy or wavelength, the phenomenon is called fluorescence. This effect can be seen if you expose a laundered white T-shirt to a black light or UV-light. The characteristic glow is caused by the fluorescence of the detergent and bleaching compounds in the shirt, which make it look white under regular light.</p>
<h4><strong>Chemiluminescence</strong></h4>
<p>In a chemiluminescent reaction, an electron is excited by absorbing heat that is released during a reaction. Then, light is released when the electron relaxes back down to the ground state. The major difference between chemiluminescence and fluorescence is that the energy that excites the electrons in chemiluminescence comes directly from the reaction.</p>
<p>One practical application of chemiluminescence is in commercial glow sticks. A glow stick contains two separate solutions: one containing peroxide and the other containing diphenyl oxalate and a color dye. When the two solutions are mixed, which occurs upon activating the glow stick, the resulting reaction between the peroxide and diphenyl oxalate produces energy that excites the dye to a higher energy state. As the reactants are exhausted, the dye returns to the ground state and releases light. This is what gives glow sticks their characteristic color.</p>
<h4><strong>Luminol</strong></h4>
<p>Luminol is a chemical that exhibits chemiluminescent properties and is utilized in a wide range of applications, most notably in forensics. Luminol (C<sub>8</sub>H<sub>7</sub>N<sub>3</sub>O<sub>2</sub>) emits blue when it is mixed with an oxidizing agent. In the case of forensics, luminol reacts with the iron in hemoglobin, enabling forensic scientists to identify very small traces of blood.</p>
<p>Luminol is synthesized by the dehydration reaction of 3-nitrophthalic acid with hydrazine. The reaction is heated to remove water, and triethylene glycol is added to further increase the temperature. The nitro group of the 3-nitrophthalhydrazide is then reduced using sodium dithionite to form an amino group at high pH. In basic conditions, the 3-nitrophthalhydrazide is soluble. The addition of glacial acetic acid precipitates the luminol.</p>
<p>With potassium hydroxide, luminol forms a dianion. The hydroxide anions deprotonate the two hydrogens that are attached to the nitrogens in the luminol. Oxygen gas oxidizes luminol to its excited state. As it relaxes back to the ground state, it releases a blue-white light.</p>
<h4><strong>References</strong></h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2015). <em>Chemistry and Chemical Reactivity.</em> Belmont, CA: Brooks/Cole, Cengage Learning.</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Concept

Synthèse du Luminol

Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. ...

UV-Vis Spectroscopy of Dyes

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Absorbance Wavelength Comparison<button class="expand">Expand</button>
	<p>In the first part of the lab, you&#39;ll use ultraviolet and visible light absorption spectroscopy, or UV-Vis absorption spectroscopy, to analyze the absorption characteristics of fluorescein, &#946;-carotene, and indigo dye. To perform UV-Vis spectroscopy, you&#39;ll place a solution between a light source and a photodetector. The molecules will absorb light at wavelengths that correspond to the energies needed to excite their electrons and scatter or transmit the rest.</p>
	<p>An absorption spectrum represents the variation in the number of photons of each wavelength that reach the detector. A higher absorbance corresponds to fewer detected photons of that wavelength. You&#39;ll take a spectrum of the pure solvent first, which the instrument will subtract from the spectrum of the dye solution to show you just the absorbance data from the dye. This reference is called a solvent blank.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you start, put on a lab coat, safety glasses, and gloves. <strong>Note: </strong>The &#946;-carotene and indigo solutions use hexane and dimethylformamide, respectively, so you will work with the dyes in a fume hood. DMF and hexane will degrade thin nitrile gloves, so change your gloves if you get solvent on them.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Now, obtain a clean quartz cuvette. Cuvettes typically have two transparent sides and two textured or opaque sides. Always hold the cuvette by the textured sides to keep the transparent sides clean.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">First, you&#39;ll analyze a solution of fluorescein in water. To make your solvent blank, fill your cuvette about 3/4 full with deionized water.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Now, clean the transparent sides of the cuvette with a laboratory wipe. Check the cuvette for air bubbles and gently tap the cuvette to dislodge them.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, bring the cuvette to the spectrometer. Open the sample chamber of the spectrometer and clean your cuvette one last time. Smudges and dust can absorb or reflect light, which will introduce errors into the data.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, insert the cuvette into the sample holder so that the transparent sides are in line with the light source. Close the sample chamber door completely. Follow the instructions for your spectrometer software to set up a scan of absorbance from 200 &#8211; 800 nm and scan your cuvette as the solvent blank.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">When the scan finishes, empty the cuvette and use compressed air to remove large drops of water.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Then, bring it to the shared hood and use a clean Pasteur pipette to fill the cuvette 3/4 full of the provided 3 &#181;M fluorescein solution.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Check the cuvette for air bubbles and remove them as needed before bringing the cuvette to the spectrometer. Remember to clean the transparent sides one last time before placing the cuvette in the sample chamber. Then, scan the cuvette as your sample.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">When the scan finishes, identify the wavelengths that fluorescein absorbed. The wavelength with the highest absorbance is called &#955;<sub>max</sub>.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Record the wavelengths of the absorbance peaks and the overall absorption range in your lab notebook and then save the data.
		<h4><strong>Table 1: Absorbance and &#955;<sub>max</sub> of dyes</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td style="text-align: center;"><strong>Absorbance range (nm)</strong></td>
					<td style="text-align: center;"><strong>&#955;<sub>max</sub> (nm)</strong></td>
				</tr>
				<tr>
					<td>&#160;Fluorescein</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>&#160;&#946;-carotene</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>&#160;Indigo</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/70/Table_1._Absorbance_and_?max_of_dyes.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, empty your cuvette into the aqueous waste and rinse it with water to remove traces of fluorescein.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">You&#39;ll analyze &#946;-carotene next, so you need a hexane solvent blank. Rinse the cuvette with hexane a few times to remove leftover water before filling it 3/4 full of pure hexane.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Remove air bubbles, clean the sides of the cuvette, and place it in the spectrometer. Make sure that the scan is set for absorbance from 200 &#8211; 800 nm, and then scan the cuvette as a solvent blank.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">When the scan finishes, empty the cuvette into the organic waste and dry it with compressed air.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Use a clean pipette to fill the cuvette 3/4 full of the provided 3 &#181;M &#946;-carotene solution and return to the spectrometer.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Clean the transparent sides of the cuvette, place it in the spectrometer, and scan it as your sample. Write the wavelengths of the peaks in your lab notebook and save the data.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, analyze the indigo dye. The indigo solution is in DMF, so empty your cuvette into the organic waste and rinse out the leftover &#946;-carotene and hexane with pure DMF.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Fill the cuvette 3/4 full of pure DMF, place it in the spectrometer, and make sure that the scan settings are the same as before. Run the solvent blank scan, and then empty the cuvette into the organic waste and dry it with compressed air.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Use a clean pipette to fill the cuvette 3/4 full of the provided 3 &#181;M indigo dye solution and scan it as your sample. Write down the absorbance information and save your data.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Lastly, empty your cuvette into the organic waste and rinse it with acetone.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">&#946;-Carotene Calibration Curve<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll use UV-Vis spectroscopy to relate absorbance intensity and concentration for &#946;-carotene by measuring the absorbances of 5 &#946;-carotene solutions with different concentrations. This will let you make a calibration curve of absorbance versus &#946;-carotene concentration and derive the equation describing that relationship.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">To begin, make a table in your lab notebook with columns for &#946;-carotene concentration and absorbance at 450 nm.
		<h4><strong>Table 2: Absorbance of &#946;-carotene</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Test </strong></td>
					<td><strong>Concentration (&#181;M) </strong></td>
					<td><strong>Absorbance at &#955;<sub>max</sub> (450 nm) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;">1.9</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;">3.7</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">7.5</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">11</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">15</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/70/Table_2._Absorbance_of_?-carotene .xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Then, get a clean cuvette and fill it 3/4 full of hexane as your solvent blank.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Remove any air bubbles, clean the transparent sides of the cuvette, and place it in the spectrometer. Set up an absorbance scan from 200 &#8211; 800 nm and scan the cuvette as the solvent blank.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">When the scan finishes, empty the cuvette into the organic waste and dry it with compressed air.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, use a clean pipette to fill the cuvette 3/4 full of the 1.9 &#181;M &#946;-carotene solution, which is the lowest concentration solution.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Clean the sides of the cuvette, insert it into the spectrometer, and scan it as a sample.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">When the scan finishes, display the absorbance intensity of the peak at 450 nm. Record this in your lab notebook as the absorbance for the 1.9 &#181;M solution.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Now, empty the cuvette into the organic waste, rinse it with hexane, and dry it with compressed air.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Use a clean pipette to fill the cuvette 3/4 full of the 3.7 &#181;M &#946;-carotene solution. Clean the sides of the cuvette, insert it into the spectrometer, and scan it as another sample.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">When the scan finishes, write down the absorbance of the peak at 450 nm. Repeat this for the remaining solutions, working from lowest to highest concentration. Save your data when you&#39;re done.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Lastly, empty the cuvette into the organic waste and clean it with acetone.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>First, look at the spectra of the three dyes. Fluorescein in water absorbs blue and purple light, with the maximum absorbance at 490 nm. It does not absorb red light, and it only absorbs some yellow and green light. Consistent with this, solid fluorescein is red, and fluorescein solutions are usually yellow to green.</p>
	<p>&#946;-carotene also absorbs blue and purple light. The maximum absorbance of &#946;-carotene in hexane is 450 nm, and you&#39;ll see another large peak at 478 nm. The strong absorption of purple light is part of why &#946;-carotene appears orange.</p>
	<p>Indigo in DMF absorbs UV light and red, orange, and yellow light, with a distinct peak at 611 nm. Thus, indigo dye reflects primarily blue and purple light, giving it its characteristic color.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-4">
		<div class="lab-step">Now, make the calibration curve for &#946;-carotene concentration. Plot the &#946;-carotene absorbance values at 450 nm from the second half of the lab against the concentrations of the solutions.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Then, apply a trend line and find the linear equation that fits the data. According to the Beer-Lambert law, in this equation, y is absorbance, x is concentration, and the slope is the product of the relevant molar attenuation coefficient and the pathlength.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Check your equation. Rearrange it to solve for concentration, and fill in the &#946;-carotene absorbance at 450 nm from the first part of the lab. The calculated concentration should be close to 3 &#181;M/L.</div>
		</li>
	</ul>
	</li>
</ol>

Ultraviolet-Visible (UV-Vis) Spectroscopy of Dyes - Procedure

Spectroscopie UV-Vis des colorants

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Absorbance Wavelength ComparisonExpand
	In the first part ...

Lab Manual Sub Articles

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Preparing 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7</strong><button class="expand">Expand</button>
	<p>In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base, disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 - 6.</p>
	<p>Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use NaOH to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before starting the experiment, calculate the mass of monosodium phosphate that you will need to make 200 mL of a 50 mM solution.
		<h4><strong>Table 1: Preparation of 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7.0</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" height="247" width="358">
			<tbody>
				<tr>
					<td colspan="2"><strong>Molar mass of NaH<sub>2</sub>PO<sub>4</sub> = 119.98 g/mol</strong></td>
				</tr>
				<tr>
					<td>Solution volume (mL)</td>
					<td style="text-align: center;">200</td>
				</tr>
				<tr>
					<td>Molarity (mM)</td>
					<td style="text-align: center;">50</td>
				</tr>
				<tr>
					<td>Moles of NaH<sub>2</sub>PO<sub>4</sub> (mol)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Mass needed (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Initial volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Final volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of NaOH used (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of DI water needed (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/45/Table_1._Preparation_of_50_mM_NaH2PO4_Buffer,_pH_7.0.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, put on the necessary personal protective equipment, including a lab coat, splash-resistant safety glasses, and gloves. <strong>Note: </strong>This section of the lab uses NaOH, which is corrosive and toxic. Use caution when pouring and transporting NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Fill a 250-mL plastic wash bottle with deionized water.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label two 100-mL beakers, one for neutral aqueous waste and one for basic aqueous waste.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Calibrate your pH meter using the provided buffers. Store the probe in its storage solution when you are done.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Tare weighing paper, and use a clean spatula to measure out the required amount of monosodium phosphate according to your calculations. Record the exact amount of monosodium phosphate in your lab notebook. <strong>Note: </strong>Monosodium phosphate absorbs moisture from the air, so work quickly to obtain an accurate measurement and close the container tightly when you are done with it.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place the monosodium phosphate in a 400-mL beaker and clean the spatula with a laboratory wipe. Throw out the weighing paper and the laboratory wipe before returning to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a graduated cylinder to measure out 175 mL of deionized water. Pour the water into the beaker of monosodium phosphate and add a magnetic stir bar.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Stir the solution until the salt has dissolved completely and the solution appears homogeneous. This usually takes 2 - 3 min.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 15 mL of deionized water and pour it into the beaker. Continue stirring the solution until it appears homogeneous again (about 1 &#8211; 2 min). Turn off the stir motor.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Rinse the pH probe with deionized water, and then clamp it in the solution with the sensor above the stir bar.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Bring a 10-mL graduated cylinder and watch glass to the dispensing hood and measure out 10 mL of 1 M NaOH. Note the exact volume in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Cover your NaOH with the watch glass, and carefully bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Resume stirring the monosodium phosphate solution. While monitoring the pH reading, use a disposable pipette to slowly add NaOH to the stirring solution in a drop-wise manner. Once the buffer pH reaches 7.0, return any NaOH still in the pipette to the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Calculate the volume of NaOH that you added to the buffer. Subtract that volume from 10 mL to determine how much deionized water you must add to your buffer to reach a total solution volume of 200 mL.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Measure the deionized water with another 10-mL graduated cylinder and add it to the stirring solution to finish making the buffer.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Rinse the pH sensor with deionized water, cover it with the storage solution filled cap, and unplug or turn off the probe.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Label a 250-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer, pH 7.0&#39;. Retrieve the magnetic stir bar from the solution.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Use a funnel to pour the buffer solution into the labeled bottle and cap it tightly.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Absorption Spectroscopy of Free and Bound Neutral Red</strong><button class="expand">Expand</button>
	<p>Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively. Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa.</p>
	<p>After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again. Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Draw the following table in your lab notebook. Number the cuvettes 1 through 10 and list the nine buffer pH values that you will use. The 10th cuvette will be a deionized water blank. <strong> Note: </strong>Always hold cuvettes by the textured sides and wipe the transparent sides just before putting the cuvette in the spectrophotometer. Remember to align the transparent sides with the beam of light in the spectrophotometer.
		<h4><strong>Table 2: Absorbance of Free and Bound Neutral Red</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Cuvette #</strong></td>
					<td style="text-align: center;"><strong>Buffer pH </strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max </sub>(Free NRH<sup>+</sup>)</strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max</sub> (Bound NRH)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;">5.0</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;">5.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">6.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">6.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">7.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">6</td>
					<td style="text-align: center;">7.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">7</td>
					<td style="text-align: center;">8.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">8</td>
					<td style="text-align: center;">8.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">9</td>
					<td style="text-align: center;">11</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">10</td>
					<td style="text-align: center;">blank</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/45/Table_2._Absorbance_of_Free_and_Bound_Neutral_Red.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Obtain ten 1.5-mL cuvettes and caps and label the caps 1 through 10 to match the table in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 25-mL beaker as &#8216;DIH2O&#8217; and fill it with deionized water.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Flush your neutral aqueous waste down the drain and relabel the beaker as &#39;aqueous buffer waste&#39;.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker for used micropipette tips.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Attach a tip to a 1-mL micropipette and use it to dispense 1000 &#181;L of deionized water into cuvette 10. This is your solvent blank.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 925 &#181;L of your pH 7.0 monosodium phosphate buffer in cuvette 5.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Bring the remaining empty cuvettes to the buffer table. Guided by the table in your lab notebook, dispense 925 &#181;L of each buffer into the appropriate cuvette using the labeled micropipettes. Be careful not to use the same pipette tip for different buffers.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once you have finished, bring the buffers back to your workbench. There, set a 200-&#181;L micropipette to dispense 75 &#181;L, and attach a tip to the micropipette.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Dispense 75 &#181;L of neutral red into each of the nine buffer cuvettes. Invert each cuvette several times to thoroughly mix the solutions. <strong>Note: </strong>Replace the pipette tip if it touches a buffer.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once you have mixed neutral red with every buffer, take a picture, or write down the colors of the solutions.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Turn on a hand-held spectrophotometer and allow the light source to warm up. Once it is ready, create a new experiment to measure absorbance versus wavelength for the free neutral red.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Insert the cuvette of deionized water and acquire a spectrum of the deionized water. Set it as a solvent background or blank.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Then, remove the blank and insert the cuvette of neutral red in the lowest pH buffer, which will have the highest concentration of protonated neutral red.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Acquire a spectrum, which should show one strong peak. Identify the wavelength corresponding to the highest point of this peak, or the maximum. Record this wavelength in your lab notebook as lambda max for protonated free neutral red.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Save the data and remove the cuvette. Record the absorbance in your notebook, then collect spectra for cuvettes 2 - 9 using the same procedure.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Record the wavelength of the isosbestic point in your lab notebook. That is where all the spectra cross at a single wavelength. If a spectrum does not cross at that point, empty and clean the cuvette, prepare a fresh sample, and try again.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Once you finish collecting spectra for all nine cuvettes, save and export the data.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Create a new experiment to measure absorbance versus wavelength for the bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Fit a new tip to the 200-&#181;L micropipette and add 75 &#181;L of riboflavin-binding protein solution to each cuvette, including the blank. Eject the tip, secure the cuvette caps, and invert the cuvette several times to mix the solutions.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Insert cuvette 10 into the spectrophotometer and set it as the solvent blank.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Acquire a spectrum of the lowest pH sample, and identify the wavelength corresponding to the maximum of the most intense peak. Record it in your lab notebook as the lambda max of protonated-bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Acquire spectra for cuvettes 2 - 9 in the same way as you did for the free neutral red samples. Record the intensities at lambda max for protonated-bound neutral red in your table, along with the wavelength at the isosbestic point.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">When you are done, save your data, export it for later analysis, and shut down the spectrophotometer.</div>
		</li>
		<li data-sub-note-item="2-27">
		<div class="lab-step">Put away all equipment and dispose of your used pipette tips in an approved waste container or trash bin.</div>
		</li>
		<li data-sub-note-item="2-28">
		<div class="lab-step">Empty the cuvettes into the aqueous buffer waste beaker and rinse the cuvettes into the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="2-29">
		<div class="lab-step">Empty the excess NaOH into the basic waste and rinse the graduated cylinder with water.</div>
		</li>
		<li data-sub-note-item="2-30">
		<div class="lab-step">Flush the basic aqueous waste down the drain with copious tap water, along with the other aqueous waste.</div>
		</li>
		<li data-sub-note-item="2-31">
		<div class="lab-step">Wash your glassware, cuvette caps, and stir bar per your lab standard procedures. Clean your work surfaces with a damp paper towel and throw out used paper towels and lab wipes in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<p>Now, let&#39;s analyze our absorbance data to determine the pKa&#39;s of neutral red.</p>
	<h4><strong>Table 3: Determination of Neutral Red pKa&#39;s</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td></td>
				<td>Free NRH<sup>+</sup></td>
				<td>Bound NRH<sup>+</sup></td>
			</tr>
			<tr>
				<td>&#160;&#955;<sub>max</sub> (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;&#916;A (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;Midpoint (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;pKa</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/45/Table_3._Determination_of_Neutral_Red_pKa.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Plot both sets of intensity data with absorbance intensity at lambda max on the y-axis and pH on the x-axis, with the points joined by smooth lines.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the difference between the starting and ending absorbance intensities for each data series.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Determine the absorbance midway between the starting and ending absorbances for each series, or the absorbance midpoints.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Find where the midpoint occurs on each line and identify the pH value at that point. These pH values are the pKa&#39;s of free and bound neutral red.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Here, we see an increase in pKa of about one between free and bound neutral red, indicating that bound-protonated neutral red is a weaker acid than free protonated neutral red by an order of magnitude.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Procedure

Tampons

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparing 50 mM NaH2PO4 Buffer, pH 7Expand
	In the first part of this experiment, you will ...

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

Dépendance à la concentration

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations of ...

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse=""><strong>Bacterial Transformation</strong>
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">IMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face away from suspension cultures, and to avoid inhaling reagents. Do not touch your face while performing the experiment. Always wash your hands before and after every experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">When you are ready to safely begin, check to make sure you have a tube or tubes of competent <em>E. coli</em> cells in your ice bucket.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, transfer 50 &#181;L from one of these tubes to each of two new micro-centrifuge tubes, one labeled '&#8211;', and one labeled '+'.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, pipet 2 &#181;L of p-GREEN plasmid into the plus tube and flick the tube gently to incorporate the plasmid throughout the bacterial solution. HYPOTHESES: The experimental hypothesis is that on plates containing no antibiotic, bacteria with or without the plasmid can survive, and so both will grow lawns of bacteria. Conversely, on the plates containing the ampicillin antibiotic, only the bacteria successfully transformed with the resistance plasmid will grow, whereas the minus plasmid plates will contain no bacteria. The null hypothesis of this experiment is that there will be no difference in bacterial growth between the plates.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Incubate both tubes on ice.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After 30 minutes submerge both tubes 3/4 of the way into the water of a 42 &#176;C water bath for 30 seconds.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">At the end of the water bath incubation, place the tubes back on ice for 5minutes, then add 950 &#956;L of room temperature SOC media to each tube.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Incubate the tubes in a shaker at 37 &#176;C for 60 minutes at 250 rpm.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">While the tubes are incubating, label the bottoms of one LB and one LB/ampicillin, or AMP plate, &#8220;+ plasmid&#8221;, and the bottoms of one LB plate and one LB/AMP plate &#8220;- plasmid&#8221;.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">At the end of the incubation, pipet 50 &#956;L from the &#8220;+ plasmid&#8221; cell tube onto both the LB/AMP plus plate and the LB plus plate.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Then, with a fresh tip, add 50 &#956;L of the bacteria that were not exposed to the plasmid to each of the minus plates.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, spread each plate using a fresh pipette tip.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Cover the plates with their lids, and then seal them with laboratory film.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then store the plates upside down in a 37 &#176;C incubator for 24 hours.
</div></li></ul></li><li data-note-item="2" data-parent-collapse=""><strong>Results</strong>
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">The day after the transformation, observe which plates have bacteria, and how the bacteria are distributed over each plate.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Record the number of colonies and the color of the colonies for different conditions. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/11/Table_1_Bacterial_Transformation.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To determine the initial mass, IM, of the plasmid, first multiply the plasmid concentration from the stock tube of p-GREEN by the volume transferred into the experimental tube.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate how much DNA is spread onto each plate, divide the volume of bacterial suspension spread onto each plate by the volume of the suspension in the tube, and multiply this fraction by the initial mass of plasmid used.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">To determine the transformation efficiency, count all of the visibly distinct colonies on the plus plasmid LB/AMP plate, and divide this number by the mass of plasmid spread. NOTE: The unit for transformation efficiencies is cfu/&#956;g of plasmid DNA.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Record all of the results for each plate in the table and analyze them.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Observe any differences between the colonies and make hypotheses to explain this observation. Consider the factors that may affect the transformation efficiency of competent bacteria.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">NOTE: You will have also noticed that each plate had a different outcome. The minus plasmid and plus plasmid LB plates should have grown a lot of bacteria across their plates. The minus plasmid LB/AMP plate should have no bacterial growth. And the plus plasmid LB/AMP plate should have grown bacterial colonies that fluoresced green.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Procedure

Transformation bactérienne

Bacterial Transformation
ExpandIMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face ...

Biologie

Génétique

Enzyme Activity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Investigating the Effect of pH and Temperature on Peroxidase Activity
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To make the extraction buffer for the peroxidase enzyme, mix equal volumes of 0.1 M sodium phosphate monobasic and 0.1 molar sodium phosphate dibasic solutions to achieve a final volume of 500 mL.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Test the resulting solution using a pH meter. The mixture should be between a pH of 6.8 and 7.2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To extract the turnip peroxidase, use a knife to remove the outer layer of a turnip and then cut the turnip into approximately two cm cubes.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place 30 g of turnip cubes into a blender with 300 mL extraction buffer, and blend the turnip cubes at high speed three times for about 15 s each time.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Filter the homogenized mixture through three layers of cheesecloth, then strain the resulting solution through one coffee filter.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Store the filtrate in a 500 mL bottle, and immediately place the bottle in the refrigerator.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, to make the enzyme substrate solutions, first add 15 mL 3% H<sub>2</sub>O<sub>2</sub> to 435 mL distilled water to dilute the H<sub>2</sub>O<sub>2</sub> to a 0.1% concentration.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To prepare the color-changing reactant, dissolve 0.2 mL concentrated guaiacol in 100 mL of isopropyl rubbing alcohol, and store this guaiacol solution in the refrigerator.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, to make solutions for the different pH conditions, add 100 mL distilled water to six 250 mL beakers, and place the buffer capsule contents with pH of 3, 4, 5, 6, 7, and 8 into separate beakers.
</div></li></ul></li></ol>

Enzyme Activity: Effect of pH and Temperature on Peroxidase Activity - Prep

Activité enzymatique

Investigating the Effect of pH and Temperature on Peroxidase Activity
ExpandTo make the extraction buffer for the peroxidase enzyme, mix equal volumes of ...

Processus cellulaires

Macromolecules

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Identifying Macromolecules with Indicator Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">HYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an indicator dye to each test solution will result in a color change if the test solution contains the macromolecule of interest. The null hypothesis might be that the addition of an indicator dye will not result in a color change in any of the test solutions.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place 16 clean test tubes in a tube rack in four rows of four tubes and label each row A through D and each column 1 - 4.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Label each test tube with its corresponding position in the test tube rack.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After this, stir solution A thoroughly and use a pipette to add 4 mL of solution A to each tube in Row A.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, stir solution B until it is well mixed and use a new pipette to add 4 mL of solution B to each tube in Row B.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After stirring solution C thoroughly, add 4 mL of solution C to each tube in Row C.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then add 4 mL of soybean-derived vegetable oil to each tube in Row D.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">When all of the tubes have been filled with their respective test solutions, add 200 mL of water to a 400 mL beaker on a hotplate and set the hotplate to high.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">To perform the Benedict&#39;s Test, add 2 mL of Benedict&#39;s reagent to test tubes A1, B1, C1, and D1 while the water is warming.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the initial color of the tubes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the water begins to boil, transfer each test tube to the beaker and heat the tubes in the boiling water bath for 3 - 5 minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, record the final color of each of the tubes in the appropriate cells of the table and turn off the hotplate. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/22/Table_1_Macromolecules.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To perform an Iodine-Potassium-Iodide Test, first observe the color of the solutions in the test tubes in column 2.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, add 1 mL of iodine potassium iodide reagent to tubes A2 to D2 and record the final color change in each of the tubes.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For the Sudan IV Test, observe and record the color of the solutions in the test tubes in column 3.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add 2 - 3 drops of Sudan IV reagent to test tubes A3 through D3 and record the color change, if any, in each tube.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">For the Biuret Test, observe the color of the solutions in the test tubes in column 4.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, add 1 mL of Biuret reagent to test tubes A4 through D4 and record the final color in each of the tubes.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Using the results from the indicator tests, each of the four original solutions can be identified as a sugar, starch, lipid, or protein. Refer to the Concepts video to help you determine which tubes contain which macromolecules.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, look at the results from the Benedict&#39;s Test. Determine which of the test solutions contain sugar based on your results.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis and the surface area in cm indicated on the y-axis.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, look at the results from the Iodine-Potassium-Iodide Test. Determine which of the test solutions contain starch.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, review the results from the Sudan IV Test. Using these observations, determine if any of the test solutions contain lipids.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Finally, look at the results from the Biuret Test. Determine if any of the test solutions contain proteins.
</div></li></ul></li></ol>

Macromolecules: Identification Using Indicator Solutions - Procedure

Macromolécules

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...