Name: ultra-high-speed western blot using immunoreaction enhancing technology
Uploaded: 2020-09-26T13:00:00.000+00:00
Duration: 5 min 59 s
Description: Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Questo lavoro è stato supportato dalla Divisione di Ricerca Intramurale, National Heart, Lung, and Blood Institute, National Institutes of Health. S.H. è stato supportato dal Japan Public-Private Partnership Student Study Abroad Program, e H.N. e K.Y. erano di Valor e V Drug Overseas Training Scholarship.

1. SDS-PAGE e trasferimento a una membrana PVDF
<ol><li>Eseguire SDS-PAGE per separare le proteine in base alle dimensioni relative. NOTA: Qualsiasi sistema di gel commerciale o fatto in casa va bene. Si prega di seguire il protocollo del produttore.</li>
	<li>Trasferire proteine separate da un gel su una membrana PVDF. NOTA: Sono adatti metodi di trasferimento comuni (trasferimento semi-secco o umido). Si prega di seguire il protocollo del produttore.</li>
</ol>2. Blocco delle membrane PVDF
<ol><li>Incubare le membrane in apposita tampone di blocco per 1 h con agitazione a temperatura ambiente. NOTA: A seconda dell'anticorpo primario e del sistema di rilevamento, deve essere selezionato un buffer di blocco appropriato. Ad esempio, i bloccanti più tipici sono BSA, latte secco non grasso, caseina e polimeri sintetici commerciali. PBS e/o TBS con 0,1% Tween20 sono i buffer più comunemente utilizzati. Questo passaggio di blocco può essere fatto durante la notte a 4 °C.</li>
</ol>3. Incubazione con anticorpo primario
<ol><li>Preparare una soluzione agente-1 per il miglioramento dell'immunoreazione al 10% (IRE-1) con acqua distillata. Vortice bene.</li>
	<li>Preparare l'anticorpo primario diluito con il 10% di IRE-1. Mescolare delicatamente e accuratamente.</li>
	<li>Se si utilizza una membrana più grande (4 cm x 8 cm - 8 cm x 8 cm), aggiungere 8 ml della soluzione anticorpale in tubi di centrifuga conica da 50 ml. Se si utilizza una membrana più piccola (2 cm x 8 cm - 4 cm x 8 cm), aggiungere 3 ml della soluzione anticorpale in un tubo di polipropilene a fondo rotondo da 14 ml(tabella dei materiali).</li>
	<li>Raccogliere la membrana PVDF con una pinzetta e drenare brevemente la soluzione di blocco. Inserire la membrana PVDF in questo tubo con le dita guantate e assicurarsi che l'intera membrana aderisca alla parete del tubo. Quando la membrana PVDF viene inserita in un tubo, affrontare il "lato proteico" della membrana che era originariamente a contatto con il gel verso l'interno. Chiudere saldamente il cappuccio.</li>
	<li>Inserire il tubo da 50 ml in una bottiglia di vetro per il forno di ibridazione. NOTA: Quando per questa incubazione vengono utilizzati tubi da 14 ml, inserire un tubo da 14 ml nel tubo da 50 mL, utilizzando un paio di supporti ad anello di plastica per mantenere la camera d'aria al centro del tubo da 50 ml.</li>
	<li>Accendere il forno di ibridazione.</li>
	<li>Incubare la membrana con rotazione di 6 giri/min per almeno 5 minuti. NOTA: Assicurarsi che la membrana aderisca alla parete in ogni momento e che il tubo (interno) sia orizzontale per coprire la membrana con la soluzione anticorpale in modo uniforme. Calibrare la diluizione dell'anticorpo e il tempo di incubazione prima dell'esperimento effettivo.</li>
</ol>4. Lavaggio a membrana
<ol><li>Interrompere la rotazione.</li>
	<li>Rimuovere con cura la membrana PVDF dal tubo con una pinzetta e posizionare la membrana in un contenitore con 50 ml di PBS-T.</li>
	<li>Risciacquare brevemente la membrana con PBS-T.</li>
	<li>Risciacquare la membrana nel contenitore con acqua distillata fino a quando non compaiono bolle. Questo per rimuovere la maggior parte dell'anticorpo.</li>
	<li>Trasferire la membrana PVDF dal contenitore allo spinner per insalate che contiene 250 mL di PBS-T.</li>
	<li>Posizionare il cestello del filtro nello spinner. Assicurarsi che il coperchio sia stato messo su in modo sicuro.</li>
	<li>Attivare lo spinner ed eseguito per circa 20-30 s.</li>
	<li>Scartare la soluzione e sciacquare brevemente l'interno dello spinner con acqua distillata.</li>
	<li>Aggiungere nuovamente 250 mL di PBS-T nello spinner.</li>
	<li>Ripetere i passaggi 4.6 e 4.7.</li>
</ol>5. Incubazione con l'anticorpo secondario
<ol><li>Preparare una soluzione agente-2 che migliora l'immunoreazione al 10% (IRE-2) con acqua distillata. Vortice bene.</li>
	<li>Preparare l'anticorpo secondario diluito con il 10% di IRE-2. Mescolare delicatamente e accuratamente.</li>
	<li>Selezionare il volume della soluzione anticorpale e del tubo come indicato al passaggio 3.3.</li>
	<li>Raccogliere la membrana PVDF con una pinzetta e scolare brevemente la soluzione. Inserire la membrana PVDF nel tubo come indicato al passaggio 3.4.</li>
	<li>Inserire il tubo da 50 ml in una bottiglia di vetro per il forno di ibridazione.</li>
	<li>Accendere il forno di ibridazione.</li>
	<li>Incubare la membrana con rotazione di 6 giri/min per almeno 5 minuti. Assicurarsi che la membrana aderisca alla parete in ogni momento e che il tubo (interno) sia orizzontale per coprire uniformemente la membrana con la soluzione anticorpale. NOTA: Calibrare il tempo di incubazione prima dell'esperimento effettivo. Quando l'anticorpo secondario è coniugato fluorescentmente, eseguire l'incubazione al buio.</li>
</ol>6. Lavaggio a membrana
<ol><li>Seguire i passaggi da 4.1 a 4.10.</li>
</ol>7. Acquisizione di immagini
<ol><li>Rilevamento chemiluminescente<ol>
<li>Posizionare un pezzo di pellicola flessibile semi-trapianto (10 cm x 15 cm) su una superficie piana.</li>
		<li>Mescolare 2 componenti del substrato chemiluminescente in un tubo da 5 ml.</li>
		<li>Trasferire immediatamente 1,5 mL del substrato misto nella pellicola flessibile semi-trapianto e posizionarla su di essa. Quindi, trasferire il resto della soluzione di substrato sulla membrana.</li>
		<li>Incubare la membrana PVDF con il substrato misto su un film flessibile semitrasparente per 1 minuto.</li>
		<li>Raccogliere la membrana PVDF con una pinzetta e drenare brevemente la soluzione del substrato.</li>
		<li>Incastro la membrana tra un paio di pellicole di trasparenza.</li>
		<li>Acquisire l'immagine in modalità chemiluminescente.</li>
	</ol>
</li>
	<li>Rilevamento fluorescente<ol>
<li>Raccogliere la membrana PVDF con una pinzetta e scolare brevemente la soluzione.</li>
		<li>Incastro la membrana tra un paio di pellicole di trasparenza.</li>
		<li>Acquisire l'immagine in modalità fluorescente. NOTA: I passaggi 7.1 e 7.2 devono essere eseguiti in prossimità della macchina per immagini per garantire la massima sensibilità.</li>
		<li>Quando il segnale di fondo è alto, eseguire una fase di lavaggio in un contenitore con 80-100 mL di PBS-T: risciacquo di 10 minuti 3 volte per l'anticorpo primario e risciacquo di 5 minuti 6 volte per l'anticorpo secondario. NOTA: Sia gli anticorpi primari che secondari diluiti con soluzioni IRE al 10% possono essere utilizzati fino a 8-10 volte senza perdita di sensibilità6. La soluzione deve essere mantenuta a 4 °C per un massimo di 1 mese.</li>
	</ol>
</li>
</ol>

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+critical+factors+for+quantitative+immunoblotting.">An analysis of critical factors for quantitative immunoblotting.</a> Science Signaling. 8 (371), (2015).</li><li>Schuck, P., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+transport+limitation+and+surface+heterogeneity+in+the+biophysical+characterization+of+macromolecular+binding+processes+by+SPR+biosensing.">The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.</a> Methods in Molecular Biology. 627, 15-54 (2010).</li><li>Li, J., Zrazhevskiy, P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Size-Associated+Diffusion+Constraints+for+Rapid+On-Surface+Bioassays+with+Nanoparticle+Probes.">Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.</a> Small. 12 (8), 1035-1043 (2016).</li><li>Higashi, 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=Old+but+not+Obsolete:+An+Enhanced+High+Speed+Immunoblot.">Old but not Obsolete: An Enhanced High Speed Immunoblot.</a> Journal of Biochemistry. 168 (1), 15-22 (2020).</li><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> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., 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=A+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. S., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+western+blot+assay+by+rotational+cyclic+draining+and+replenishing+procedure.">Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.</a> Electrophoresis. 39 (23), 2974-2978 (2018).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+other+notable+protein+detection+methods+on+blots.">A brief review of other notable protein detection methods on blots.</a> Methods in Molecular Biology. 536, 557-571 (2009).</li><li>Nagase, H., 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=Reliable+and+Sensitive+Detection+of+Glycosaminoglycan+Chains+with+Immunoblots.">Reliable and Sensitive Detection of Glycosaminoglycan Chains with Immunoblots.</a> Glycobiology. , (2020).</li></ol>

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Western blot è una tecnica analitica ampiamente utilizzata per rilevare proteine specifiche che è stata sviluppata ~ 40 annifa 7,8. Da allora, la tecnica ha continuato ad evolversi, con successive innovazioni che migliorano la sensibilità, la velocità e la quantificazionedella tecnica 2,9,10,11,12,...

Una macchia occidentale (nota anche come immunoblot) è una tecnica potente e fondamentale in un'ampia gamma di discipline scientifiche e cliniche. Questa tecnica è usata per esaminare la presenza, l'abbondanza relativa, la massa molecolare relativa e le modifiche post-tras traslizionali delle proteine1. Combinato con l'analisi digitale delle immagini, questo metodo può analizzare in modo affidabile l'abbondanza di proteine e modificheproteiche 2. Sebbene la macchia occidentale sia eseguita regolarmente, è un metodo dispendioso in termini di tempo e manodopera. Sono necessarie lunghe incubazioni dell'anticorpo con membrane. Qui descriviamo una modifica del metodo di incubazione che supera questa limitazione senza sacrificare la sensibilità.
Durante l'incubazione della membrana, gli anticorpi galleggiano in soluzione mentre gli antigeni vengono immobilizzati sulla membrana. A causa della loro elevata affinità, il tasso di legame degli anticorpi all'antigene è più veloce della diffusione degli anticorpi dalla soluzione sfusa alla membrana. In questo modo si crea un "livello di esaurimento" a bassa concentrazione (Figura 1). Potrebbero essere necessario ore prima che anticorpi più distanti raggiungano la membrana attraverso la diffusione passiva, che è il principale fattore responsabile dei lunghi tempi di incubazione in questa tecnica. Questo effetto è chiamato limitazione del trasporto di massa (MTL)3. È stato proposto che il drenaggio ripetitivo e il rifornimento della soluzione contenente anticorpi possano interrompere lo strato di esaurimento e superare l'MTL4. Qui, abbiamo ideato una tecnica unica che implementa questo concetto ciclico di drenaggio e rifornimento (CDR) per diminuire l'effetto dell'MTL in un tradizionale protocollo di soffiatura occidentale e ridurre notevolmente il periodo di incubazione richiesto.
Al fine di mantenere la sensibilità di rilevamento con un breve tempo di incubazione, abbiamo fatto uso di una tecnologia di miglioramento dell'immunoreazione che accelera la reazione antigene-anticorpo, migliorando così il rapporto segnale-rumore5. Molti agenti di miglioramento dell'immunoreazione disponibili in commercio (IRE) sono costituiti da 2 componenti (soluzione 1 e 2, vedi Tabella dei materiali). La composizione proprietaria o il meccanismo d'azione dell'IRE non è stato divulgato, ma abbiamo precedentemente scoperto che IRE ha diminuito la costante di dissociazione tra l'antigene e l'anticorpo nei test di legame in fase solida, indicando che una maggiore affinità è, almeno in parte, responsabile dell'effetto di miglioramento dell'IRE6. La combinazione di CDR e IRE produce un protocollo western blotting ad altissima velocità che riduce l'intero tempo di procedura senza sacrificare la sensibilità.

<table><tbody><tr><td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>Apollo Transparency Film for Laser Printers</td><td>N/A</td><td>N/A</td><td>Any kinds of Laser Printer Transparency Film are fine.</td></tr><tr><td>Azure Imaging System 600</td><td>Azure Biosystems</td><td>Azure 600</td><td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td></tr><tr><td>Can Get Signal Immunoreaction Enhancer Solution</td><td>TOYOBO</td><td>NKB-101</td><td></td></tr><tr><td>CARNATION Instant Nonfat Dry Milk</td><td>Carnation</td><td>N/A</td><td></td></tr><tr><td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from sheep</td><td>GE Healthcare</td><td>NA9310V</td><td></td></tr><tr><td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from donkey</td><td>GE Healthcare</td><td>NA9340V</td><td></td></tr><tr><td>HYBAID Micro-4</td><td>N/A</td><td>N/A</td><td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td></tr><tr><td>Immobilon-P PVDF Membrane, 0.45 &amp;micro;m pore size</td><td>Millipore Sigma</td><td>IPVH304F0</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td><td>LICOR</td><td>926-68070</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td><td>LICOR</td><td>926-32211</td><td></td></tr><tr><td>KPL LumiGLO Reserve Chemiluminescent Substrate</td><td>seracare</td><td>5430-0049</td><td></td></tr><tr><td>Mouse anti-&amp;szlig; actin antibody</td><td>Millipore Sigma</td><td>A5316</td><td></td></tr><tr><td>Odyssey Blocking Buffer (PBS)</td><td>LICOR</td><td>927-40100</td><td>Blocking Buffer for fluorescent detection</td></tr><tr><td>OXO Salad Spinner</td><td>OXO</td><td>32480V2B</td><td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td></tr><tr><td>Parafilm Sealing Film</td><td>Bemis</td><td>Parafilm M PM996</td><td>semi-transparent flexible film</td></tr><tr><td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Rings to hold 14 ml tube in the center of 50 ml tube</td><td>N/A</td><td>N/A</td><td>Prepared in a machine shop</td></tr><tr><td>Rabbit anti-6X His tag antibody</td><td>Abcam</td><td>ab9108</td><td></td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>P2287</td><td></td></tr></tbody></table>

ultra-high-speed western blot using immunoreaction enhancing technology

Una macchia occidentale (nota anche come immunoblot) è un metodo canonico per la ricerca biomedica. È comunemente usato per determinare la dimensione relativa e l'abbondanza di proteine specifiche e le modifiche delle proteine post-traslazionali. Questa tecnica ha una ricca storia e rimane ampiamente utilizzata per la sua semplicità. Tuttavia, la procedura di gonfiore occidentale richiede notoriamente ore, anche giorni, per essere completata, con un collo di bottiglia critico che sono i lunghi tempi di incubazione che ne limitano la produttività. Questi passaggi di incubazione sono necessari a causa della lenta diffusione degli anticorpi dalla soluzione sfusa agli antigeni immobilizzati sulla membrana: la concentrazione di anticorpi vicino alla membrana è molto inferiore alla concentrazione di massa. Qui presentiamo un'innovazione che riduce drasticamente questi intervalli di incubazione migliorando il legame dell'antigene attraverso il drenaggio ciclico e il rifornimento (CDR) della soluzione anticorpale. Abbiamo anche utilizzato una tecnologia di miglioramento dell'immunoreazione per preservare la sensibilità del saggio. Una combinazione del metodo CDR con un agente di miglioramento dell'immunoreazione commerciale ha aumentato il segnale di uscita e ridotto sostanzialmente il tempo di incubazione degli anticorpi. La macchia occidentale ad altissima velocità risultante può essere eseguita in 20 minuti senza alcuna perdita di sensibilità. Questo metodo può essere applicato alle macchie occidentali utilizzando sia il rilevamento chemiluminescente che fluorescente. Questo semplice protocollo consente ai ricercatori di esplorare meglio l'analisi dell'espressione proteica in molti campioni.

Questo esempio illustra l'efficacia del metodo CDR insieme alla tecnologia di immunoenhancing sulla macchia occidentale. L'incubazione statica è stata eseguita su un film flessibile semitrasparente in cui le membrane PVDF sono state incubate con la soluzione anticorpale, mentre l'incubazione CDR era in un forno di ibridazione da tubi rotanti che contenevano le membrane e la soluzione anticorpale (Movie). La figura 2 ha mostrato quantità di antigene e anticorpo, rispettivamente, necessarie ...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Una tecnica di soffiatura occidentale ad altissima velocità viene sviluppata migliorando la cinetica del legame antigene-anticorpo attraverso la tecnologia di drenaggio e rifornimento ciclico (CDR) in combinazione con un agente che migliora l'immunoreazione.

Western Blot ad altissima velocità che utilizza la tecnologia di miglioramento dell'immunoreazione

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

5.8K Views.  National Institutes of Health. Una tecnica di soffiatura occidentale ad altissima velocità viene sviluppata migliorando la cinetica del legame antigene-anticorpo attraverso la tecnologia di drenaggio e rifornimento ciclico (CDR) in combinazione con un agente che migliora l'immunoreazione.

Video: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Un protocollo ottimizzato per elettroforetica Mobility Shift Assay Utilizzando oligonucleotidi infrarosso colorante fluorescente etichettati

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Ricerca

Biochimica

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting Utilizzando il Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Biologia

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Rilevamento altamente sensibile e quantitativo delle proteine e loro isoforme dal metodo di messa a fuoco isoelettrica capillare

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Analisi immunoblot

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blotting: la preparazione dei campioni di rilevamento

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

Il più veloce occidentale in città: un tocco contemporaneo alla classica analisi Western Blot

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

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 within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

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.

Una guida al moderno Quantitative Fluorescent Western Blotting con strategie di risoluzione dei problemi

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 ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Tecniche basate sull'immuno-precipitazione: purificazione di proteine endogene con l'impiego di microsfere di agarosio

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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Biologia avanzata

Immunologia

The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

Il western blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

Biologia di base

Metodi di base in biologia cellulare e molecolare

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Saggio di Western Blot chemiluminescente per la lavorazione delle proteine

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...