Name: a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation
Uploaded: 2017-03-27T14:00:00.000+00:00
Duration: 6 min 34 s
Description: Watch this Scientific Journal Video about A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation at JoVE.com

Yazarlar teknik yardım için Agilent teşekkür etmek istiyorum. Kate Markham ve Rafael Palomino Dr Hsiau-Wei Lee yazının Şekil 1 hazırlanmasında yaptığı yardım için teşekkür edilmektedir Aβ peptid ve onların ilk sentezinde yardım ve arıtma için yatırılır.

Aβ40 ya Aβ42 Peptid 1. Hazırlayıcı HPLC saflaştırma <ol><li> HPLC saflaştırması için, aşağıdaki tamponlar hazırlamak. <ol><li> Ultra saf su 1000 ml NH4OH (% 28 çözelti) 1.3 mL ilave edilerek tampon A (20 mM NH4OH) hazırlayın. </li><li> HPLC-grade asetonitril 800 mL ve ultra saf su 200 mL çözeltisine NH4OH (% 28 çözelti) 1.3 mL ilave edilerek tampon B (20 mM NH4 OH ile% 80 asetonitril) hazırlayın. </li><li> Ultra saf su ve 100 mL NH4OH (% 28 çözelti), 100 uL eklenmesiyle Örnek erime tamponu (% 0.1 NH4OH) hazırlayın. </li></ol></li><li> Kurulum, Şekil 1 &#39;de gösterildiği gibi, HPLC aleti. <ol><li> A tampon ve polimer tüp kullanarak HPLC pompası girişlerine B tampon içeren çözücü şişe yerleştirin. tek parça uydurma HPLC pompası polimer boru takın. polimer boru emin olunher bir tampon aygıtının doğru giriş valfı takılmıştır. Bir degazör ile HPLC pompası takın. </li><li> Çift 300 Å 8 mikron 25 mm х 300 mm hazırlayıcı sütun girişi ile HPLC pompası polimer boru kullanılarak (Şekil 1B, aşırı sol sütun, Malzeme Listesi bakınız). <ol><li> tek parça parmak sıkı geçme ile hazırlayıcı sütun polimer boru takın. Polimer sütunu doğru bir şekilde yönlendirilmesini sağlamak. Not: Hazırlama sütunun durağan faz poli (stiren-divinilbenzen) parçacıklar oluşur. Polimer kolonun doğru yönelim tek yönlü ok ile dış gövde üzerinde işaretlenir. </li></ol></li><li> Polimer boru kullanarak ikili dalga boyu dedektörü sütunun çıkışını bağlayın ve 214 nm ve 280 nm dalga boyu dedektörü ayarlayın. <ol><li> kurulum enstrüman yöntemi seçeneği belirleme dalgaboyu parametrelerini değiştirerek dalga boyu AlterDahili HPLC yazılımı. </li></ol></li><li> tek parçalı bir parmak sıkı geçme ile bir dalga boyu detektörü girişine polimer boru takın. dalga boyu dedektörü çıkış vanasına polimer boru takın. tek parça parmak sıkı geçme ile HPLC dedektörü çıkış vanasına polimer boru takın. Bu örnek toplama tüp olacaktır. Not: Aβ peptidi üzerinde güçlü bir kromofor eksikliği 214 nm pik toplama primer ultraviyole (UV) bir dalga boyu olarak kullanılması zorunlu kılar. </li></ol></li></ol><img alt="Şekil 1" src="/files/ftp_upload/55482/55482fig1.jpg" /> Şekil 1: beta amiloid peptidlerin saflaştırılması için kullanılan HPLC aygıtının deney düzeneği. (A) bir gaz giderici ve 214 nm ve 280 nm ayarlı değişken dalga uzunluğu detektörü ile donatılmış kuaterner HPLC pompası; (B) olarak hazırlanmıştır HPLC sütunları arasında, beta amiloid peptidlerin saflaştırılması için kullanılansoldan sağa, 25 x 300 mm, 2, hazırlayıcı kolon, 7.5 x 300 mm, 2, yarı preparatif kolonu ve 4.6 x 250 mm 2 Analitik kolon; (C) analitik HPLC için kullanılan 20 uL paslanmaz çelik enjeksiyon döngü ile Manuel enjektör; (D) hazırlayıcı ve yarı hazırlayıcı saflaştırma için kullanılan 10 ml paslanmaz çelik enjeksiyon döngü ile manuel enjektör. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <ol start="3"><li> (HPLC yazılımı yerleşik kurulum enstrüman yöntemi seçeneği) çözücü tarife parametresini değiştirerek arıtma yöntemi giriniz Tablo 1&#39;de görüldüğü gibi arıtma yöntemini çalıştırmak için HPLC yazılım programı. HPLC yazılım üzerinde &quot;açık&quot; butonuna tıklayarak HPLC pompası açın. NOT: Pompa tampon A başlangıç ​​oranını temin başlar ve hazırlık yoluyla B tampon olacakarative kolon ve HPLC aleti. <ol><li> 30 dakika tamamen dengelenmesi için sistem bırakın. </li></ol></li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> Saat / dakika </td><td> Tampon A% &#39;si </td><td> Tampon B b% </td><td> Akış Hızı d / ml dak -1 </td></tr><tr><td> 0 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 45 </td><td> 75.5 </td><td> 24.5 </td><td> 6 </td></tr><tr><td> 45.01 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 52.01 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 52.02 </td><td> 73 </td><td> 27 </td><td> 6 </td></tr><tr><td> 85 </td><td> 73 </td><td> 27 </td><td> 6 </td></tr><tr><td> 92 </td><td> 5 </td><td> 95 ° C </td><td> 6 </td></tr></tbody></table> &lt;strong&gt; Tablo 1: 25 × 300 mm polimer kolon kullanılarak Aβ42 ve Aβ40 peptidler saflaştırılması için zaman çizelgesi. Bir Tampon A - 20 mM NH4 OH ile H2O; 20 mM NH4 OH ile B% 80 MeCN /% 20 H2O; örnek aşağıdaki enjeksiyondan önce sütun yıkama 15 dakika Cı Ran; D HPLC enstrümantasyon 6 mL / dakika bir akış hızı çalıştırmak için, basınç sınırı 200 bar azaltılması gerekir. <ol start="4"><li> Aβ peptidi numunenin saflaştırılması Not: Ham peptid bir otomasyonlu katı faz peptid sentezi ile elde edildi. 10 <ol><li> Örnek erime 4 ml tampon ham Aβ peptidi 3 mg eritin. Oda sıcaklığında 30-60 sn ve çözünmeye yardımcı olması için 40 kHz frekansta örnek sonikasyon. </li><li> 16-gauge ile donatılmış 5 mL&#39;lik bir plastik bir şırınga kullanılarak HPLC kolonuna Numunenin tamamı enjektepaslanmaz çelik iğne. alt aşama 1.3 belirtildiği gibi arıtma yöntemi çalıştırın. NOT: Sistem numune enjeksiyon 10 ml paslanmaz çelik enjeksiyon döngüsü (Şekil 1D) ile donatılmış bir el enjektörün kullanımı ile yapılabilir sağlar. İstenen Aβ peptid keskin çözülmesi tepe (Şekil 2C) olarak 72 ila 74 dakika Zehir. </li><li> 50 ml konik santrifüj tüpüne örnek toplamak. Toplanan eluent doğrudan enjeksiyon kütle spektrometresi ile Aβ zirve kimliğini onaylamak. 11 -20 ° C&#39;de en fazla 12 saat boyunca yıkama sıvısı. NOT: daha uzun 12 saat peptid oksidasyonu için potansiyeli nedeniyle tavsiye edilmez dönemler için çözümün depolama. </li><li> Toplanan kısım dondurma flaş saflaştırılmış peptid Yalıtmak / sıvı azot ve Lyophilize içinde Aβ peptid alikotları. -60 ° C ve bir pres içinde bir sıcaklıkta dondurularak kurutulması örnek tarafından liyofilizasyon gerçekleştirmek24 saatlik bir süre için 20 mTor olun. </li><li> Aβ peptidi saflığını belirlemek için aşağıda verilen analitik HPLC protokolü çalıştırın. 6 aya kadar bir süre için -20 ° C &#39;de bunların liyofilize formda saklayın peptidler. </li></ol></li></ol> Saflaştırılmış Aβ Proteininin 2. Analitik HPLC Analizi <ol><li> alt bölümde 1.1 belirtildiği gibi HPLC tamponlar hazırlayın. Yukarıdaki protokol. </li><li> Kurulum aşama 1.2.1 uyarınca ve 4.6 x 250 mm analitik sütun (Şekil 1B, en sağ sütun) ve 20 uL, paslanmaz çelik enjeksiyon döngüsü ile el enjektör ile, Şekil 1 &#39;de gösterildiği gibi (Şekil 1C) monte analitik HPLC Enstrüman. </li><li> Adım 1.3 ile benzer talimatları izleyerek Tablo 2&#39;de görüldüğü gibi analitik yöntemini çalıştırmak için HPLC yazılım programı. </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> Zaman /dk </td><td> Tampon A% &#39;si </td><td> Tampon B b% </td><td> Hızı / ml dak akış -1 </td></tr><tr><td> 0 </td><td> 95 </td><td> 5 </td><td> 1 </td></tr><tr><td> 30 </td><td> 50 </td><td> 50 </td><td> 1 </td></tr></tbody></table> Tablo 2: Aβ peptidi HPLC saflığı analizi için takvim. Bir Tampon A - 20 mM NH4 OH ile H2O; 20 mM NH 4 OH b% 80 MeCN /% 20 H 2 O. <ol start="4"><li> Aβ peptidi saflığı analizi <ol><li> Örnek tampon çözeltisi içinde peptidin çözülmesi yoluyla saflaştırılmış peptidin 1 mg / ml çözelti hazırlayın. NOT: Tampon tarifi alt bölümde 1.1 bulunabilir. Protein konsantrasyonu, 280 nm&#39;de (A 280nm) protein emiliminin ölçülmesi ile belirlenir. 12 m<html> konsantrasyonunu belirlemek için kullanılır olar yok olma katsayısı (ε) ε = 1.490 dm3 mol-1 cm-1 olan. 13 </li><li> HPLC kolonuna 1 mg / ml (222 uM) çözeltisinin 20 ul enjekte edilir ve adım 2.3 kurulum oldu analitik yöntem çalıştırın. Not: analiz için kullanılmaz kalan çözelti, sıvı nitrojen içinde dondurulur ve Aβ peptidi kurtarmak için liyofilize edilebilir. Liyofilizasyon ayrıntıları alt bölümde 1.4.4 bulunabilir. Aβ peptidi 16 ve 18 dakika boyunca (Şekil 2B) arasındaki analitik sütundan elute olur. Aβ peptidi saflığını belirlemek için HPLC aleti birlikte yerleşik entegre analiz yazılımı kullanarak. Saflık spektrumunun bireysel zirveleri her entegre ve peptit tepe yüzdesi alanını hesaplanarak belirlenir. Tipik olarak,&gt;% 95 bir saflaştırma saptanmalıdır. </li></ol></li></ol><img alt="&lt;/ Html">&quot;Şekil 2&quot; src = &quot;/ files / ftp_upload / 55482 / 55482fig2.jpg&quot; /&gt; Şekil 2: Aβ42 Temsilcisi HPLC izleri. (A) Geleneksel C4 silika saflaştırma koşulları: Tampon A: H2O,% 0.1 trifluoroasetik asit (TFA), B tampon maddesi ile:% 0.1 TFA, gradyan ile MeCN (asetonitril): 20 tampon B 40 dakika boyunca,% 27 ve ardından izokratik% 27 B tamponu ile; (B) 25 x 300 mm, 2 Polimer kolonu, koşullar kullanılarak hazırlayıcı saflaştırma: 20 mM NH4OH, tampon B ile H2O: Tampon A% 80 MeCN /% 20, H, 20 mM NH4 OH ile 2, O, gradyan : izokratik% 27 tampon B, ardından 70 dakika boyunca% 20 ila 27 tampon B; 25 x 300 mm, 2 Polimer kolonu kullanılarak hazırlayıcı saflaştırmaya Optimize (C) koşulları alt bölüm protokolü açıklama metni 1.3 bulunan Tablo 1 &#39;de tarif edilir; (D) Analitik HPLC 4.6 x 250 mm 2 Polimer kolonu, kon kullanılarakitions: Tampon A: H2O, 20 mM NH4OH, tampon B ile: 30 dakika boyunca 20 mM NH4OH, gradyan% 5 ila 50 tampon B% 80 MeCN /% 20 H2O. Bölüm A, B ve C Aβ42 tekabül zirve bir yıldızla işaretlenir. Bölüm D. Kütle spektrometrisi gösterildiği gibi C&gt;% 95 bir saflığa ortaya kısmen işaretlenmiş Aβ42 tepe toplama Aβ42 zirvesinin ne olduğu belirlemek için kullanılmıştır. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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Yazarlar ifşa hiçbir şey yok.

Aβ peptidi HPLC saflaştırma arıtılmadan kullanılan durağan faz peptid elüt edilmesi için seçilen mobil faz iki seçimi oldukça bağlıdır. toplama peptidin yüksek eğilimi düşük izoelektrik noktası Aβ peptidi uzun bir geniş olmayan çözüldü olarak elüsyon ile hidrofobik proteinlerin ayrılması (C4 veya asidik bir mobil elüsyon ile birleştirilmiş C8 faz) zor geleneksel kromatografik koşullan sağlamaya pik (Şekil 2A). Yüksek pH değerinde kimyasa...

Alzheimer hastalığı bir nörodejeneratif bir hastalıktır etkileri 35 milyondan fazla kişi dünya çapında. Hastalığın başlangıcı ve gelişiminde güçlü Belirtilmiş 1, yüksek toplama eğilimli, hidrofobik peptid Amiloid beta (Aβ) &#39;dir. 2 Aβ Ancak, beta amiloid 42 (Aβ42), proteinin en toksik bir şekilde 42-amino asit türevi olduğu düşünülmektedir, uzunluğu 36 ila 43 amino asit arasında değişir. 3 Bu hali hazırda, özellikle nörotoksik maddeler olduğu düşünülmektedir yayılabilir oligomerik türler oluşturma Aβ42 yeteneğini büyük bir kısmında kaynaklanmaktadır. 4 Aβ peptid anlayışımızı ilerletmek için, rutin yüksek saflıkta malzeme elde etmek esastır. saf olmayan artıklarının varlığı büyük ölçüde peptidin birikme eğilimi özelliklerini değiştirmek için gösterilmiştir. 5 Traditionally, örneğin Aβ hidrofobik peptitler, yüksek performanslı sıvı kromatografisi (HPLC) ayırma C 4 ya da Cı 8 silis bazlı sabit fazlarda ve bir asidik mobil faz bir kombinasyonunun kullanımı ile yapılmıştır. 6 Ancak, bu tür koşullar peptid saflaştırılması için bir meydan okuma sunabilir. Aβ peptidi düşük izoelektrik noktası (pl yaklaşık 5.5) 7, asidik koşullar altında, peptid agregasyon artar ve bunun bir sonucu olarak genellikle izole etmek zordur, geniş olmayan çözüldü HPLC tepe (Şekil 2A) üretilmeleri anlamına gelir. Ayrıca, bu tür geniş zirveler genellikle peptid toplama profilini etkileyebilecek ve yaygın dramatik üretilen peptid miktarını etkileyebilir arınma sonraki mermi gerektirebilir safsızlıklar içermezler. poli (stiren / divinilbenzen) durağan faz, PLRP-S, saflaştırmadan bir alternatif araçları temsil ederYing hidrofobik peptitler. hareketsiz faz farklı protein ve haberci ribonükleik asitlerin (mRNA) &#39;in bir dizi saflaştırılmasında kullanılmıştır. 8, 9 PLRP-S sabit faz ters faz ayrılması için ek bir alkil ligandı gerektirir ve daha da önemlisi, peptidin agregadan arındırma çaresinin yol açan yüksek pH değerinde kimyasal olarak kararlıdır. 7 Burada, yüksek saflıkta amiloid beta 42 (Aβ42) ve amiloid beta 40 (Aβ40) peptidleri veren bir özel HPLC arıtma protokolü sunduk.

<table><tbody><tr><td>Agilent 1260 Infinity II quarternary pump</td><td>Agilent</td><td>G7111B</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump</td></tr><tr><td>Agilent 1260 Infinity II Dual variable wavelength detector</td><td>Agilent</td><td>G7114A</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop</td><td>Agilent</td><td>0101-1232</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 20 &amp;micro;L stainless steel sample loop</td><td>Agilent</td><td>G1328C</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Ring Stand Mounting Bracket</td><td>Agilent</td><td>1400-3166</td><td></td></tr><tr><td>Agilent PLRP-S 300 &amp;Aring; 5&amp;nbsp;&amp;micro;m 4.6 x 250 mm (Analytical)</td><td>Agilent</td><td>PL1512-5501</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td></tr><tr><td>A&amp;beta;42 or A&amp;beta;40 peptide</td><td></td><td></td><td>Synthesized in-house using a CEM liberty automated peptide synthesizer.</td></tr><tr><td>Ammonium Hydroxide (NH&lt;sub&gt;4&lt;/sub&gt;OH, 28% solution)</td><td>Fisher Scientific</td><td>A669-500</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>A998-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W5-4</td><td></td></tr><tr><td>Falcon 50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>14-954-49A</td><td></td></tr><tr><td>Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea</td><td>Sigma-Aldrich</td><td>Z227250</td><td></td></tr><tr><td>Normject 5&amp;nbsp;cc sterile syringe</td><td>Fisher Scientific</td><td>1481729</td><td></td></tr><tr><td>16 Gauge SS Needle</td><td>Rheodyne</td><td>3725-086</td><td></td></tr></tbody></table>

a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

PLRP-S durağan faz bir kombinasyonunu 72 ve 74 dakika boyunca (Şekil 2C) arasında bir alıkonma zamanında Aβ peptidi için keskin çözüldü tepe oluşumunda yüksek bir pH mobil faz sonuçları kullanılarak Aβ42 peptidinin arıtılması. tepe kimliğinin teyidi toplanan elüsyon direkt enjeksiyonu kütle spektrometrisi yoluyla yapılır. yıkama sıvısı kadar 12 saat boyunca çözelti içinde -20 ° C&#39;de muhafaza edilebilir. depolamanın daha uzun süreli ...

Watch this Scientific Journal Video about A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation at JoVE.com

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Bu yazıda oligomer oluşturabilen yüksek saflıkta amiloid beta 42 (Aβ42) ve amiloid beta 40 (Aβ40) peptidler, veren bir özel HPLC arıtma protokolü sunduk. Amiloid P Alzheimer hastalığında rol yüksek çekiş eğilimli hidrofobik peptittir. peptid amiloidojenik doğası onun saflaştırma bir meydan okuma yapar.

Bir Ismarlama HPLC Arıtma Protokolü That Oligomer Formasyonu Yetenekli Verim Yüksek saflıkta Amiloid Beta 42 ve Amiloid Beta 40 peptidler,

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

a-tailored-hplc-purification-protocol-that-yields-high-purity-amyloid

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.8K Görüntüleme.  University of California, Santa Cruz.  Bu yazıda oligomer oluşturabilen yüksek saflıkta amiloid beta 42 (Aβ42) ve amiloid beta 40 (Aβ40) peptidler, veren bir özel HPLC arıtma protokolü sunduk. Amiloid P Alzheimer hastalığında rol yüksek çekiş eğilimli hidrofobik peptittir. peptid amiloidojenik doğası onun saflaştırma bir meydan okuma yapar. 

Video: A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Β-amiloid Oligomerik hazırlanması 1-42</subHipokampal Dilimleri Sinaptik Plastisite değer düşüklüğü> İndüksiyon

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Nörobilim

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Amiloid beta-Protein, Alzheimer hastalığının etkeni Aptamers Seçimi

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

A11-pozitif β-amiloid Oligomer hazırlama ve değerlendirme sistemini kullanarak Dot Blot Analizi

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Biyokimya

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amiloid prekürsör protein intrasellüler Domain saflaştırılması ve toplama

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Tıp

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine tabanlı peptid Amphiphiles (PPAs) ve ilgili Biyomalzeme kendi kendine montaj sentezi için facile iletişim kuralı

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Kimya

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

1,2-Dithiolane malzemelerin ve kendi kendine montaj peptidler değiştirilme tarihi

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Amiloid β42 toksisite ve ateş yetişkin Zebra balığı beyinde modelleme

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

Amiloid Beta 1-40 Kaplı Altın Kolloidlerin pH'a Bağlı Tersinir Kendi Kendine Montajının Karakterizasyonu

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biyoloji

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Beyinden Amiloid Fibril Çekirdeklerinin Biyokimyasal Saflaştırılması ve Proteomik Karakterizasyonu

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Bir Ismarlama HPLC Arıtma Protokolü That Oligomer Formasyonu Yetenekli Verim Yüksek saflıkta Amiloid Beta 42 ve Amiloid Beta 40 peptidler,

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Β-amiloid Oligomerik hazırlanması_{1-42 İndüksiyon}

Amiloid beta-Protein, Alzheimer hastalığının etkeni Aptamers Seçimi

A11-pozitif β-amiloid Oligomer hazırlama ve değerlendirme sistemini kullanarak Dot Blot Analizi

Amiloid prekürsör protein intrasellüler Domain saflaştırılması ve toplama

Polyamine tabanlı peptid Amphiphiles (PPAs) ve ilgili Biyomalzeme kendi kendine montaj sentezi için facile iletişim kuralı

1,2-Dithiolane malzemelerin ve kendi kendine montaj peptidler değiştirilme tarihi

Amiloid β42 toksisite ve ateş yetişkin Zebra balığı beyinde modelleme

Amiloid Beta 1-40 Kaplı Altın Kolloidlerin pH'a Bağlı Tersinir Kendi Kendine Montajının Karakterizasyonu

Rapid Generation of Amyloid from Native Proteins In vitro

Beyinden Amiloid Fibril Çekirdeklerinin Biyokimyasal Saflaştırılması ve Proteomik Karakterizasyonu

Bir Ismarlama HPLC Arıtma Protokolü That Oligomer Formasyonu Yetenekli Verim Yüksek saflıkta Amiloid Beta 42 ve Amiloid Beta 40 peptidler,

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Amiloid prekürsör protein intrasellüler Domain saflaştırılması ve toplama

Polyamine tabanlı peptid Amphiphiles (PPAs) ve ilgili Biyomalzeme kendi kendine montaj sentezi için facile iletişim kuralı

1,2-Dithiolane malzemelerin ve kendi kendine montaj peptidler değiştirilme tarihi

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Rapid Generation of Amyloid from Native Proteins In vitro

Beyinden Amiloid Fibril Çekirdeklerinin Biyokimyasal Saflaştırılması ve Proteomik Karakterizasyonu

Β-amiloid Oligomerik hazırlanması_{1-42 İndüksiyon}