Yazarlar AH Yaşam ödülü için UF Bilim Howard Hughes Tıp Enstitüsü teşekkür

1. Kasetleri hazırlanıyor <ol><li> Temiz ön ve arka kullanılmış Invitrogen Nupage Novex minigel kaset, veya diğer ticari minigel kaset plakaları. </li><li> Plastik epoksi yaklaşık 1-3 ml hazırlayın ve üreticinin talimatlarına göre iyice karıştırın. </li><li> Kaset monte edildiğinde iki tabak temas edecek ön plaka, iç kenarına epoksi uygulayın. </li><li> Açma kapama aşırı epoksi önlemek için kaset montaj sırasında arka yüzeyinin alt yarık aracılığıyla filtre kağıdı ya da bir karton parçası kaydırarak. Kısa sürede iki tabak monte gibi filtre kağıdı çıkarın. </li><li> Sıkı bir mühür için kenarları boyunca bağlayıcı klipleri yerleştirin ve kasetleri tamamen tedavi etmek için epoksi gece bekletin. </li><li> Elektrik bandı ile arka plaka altındaki yarık sürün. </li></ol> 2. Çözme jel Dökme <ol><li> 125 ml yan kol şişesi için aşağıdaki ekleyin:4X Tris-bazlı Ayrıştırıcı jel tampon pH 8.70,% 30.8 akrilamid / bisacrylamide 3.50 ml, Tip 3.25 ml, 2.25 ml 9 ml &#39;lik bir toplam hacim tepkin madde gradlı I için su (IRG-H2O). </li><li> De-gaz vakum aparatı şişenin yan kol bağlama ve şişenin üstüne ters bir stoper yerleştirerek 10 dakika bir çözüm. </li><li> Bir 50 ml tek kullanımlık polipropilen santrifüj tüpüne de-gassed çözeltisi 9 ml aktarılır ve 30% 10 ammounium persülfat (APS) ile ul ve N ul 6, N, N &#39;, N&#39;,-tetramethylethylenediamine (TEMED) eklenir ve karıştırılır kabarcıkları tanıtma önlemek için çözüm dönen tarafından iyi. </li><li> Hemen jel plakları arasında meydana gelen herhangi bir kabarcıklarını engellemek için kaset tarafı aşağı çalışmasına izin, kaset içine çözüm dökün. Ön (daha kısa) plakanın üst kenarından yaklaşık 2 cm ile kaset doldurun. </li><li> Yavaşça 1X çözülmesi jel tamponu ile doyurulmuş 1-bütanol ve 0.5 ml ilave jel 1 için polimerize izinsaat. </li></ol> 3. Istifleme jel Dökme <ol><li> 4X Tris-bazlı bir istifleme jel tampon pH 6.80,% 30.8 akrilamid / bisacrylamide 0.40 ml, 3 ml &#39;lik bir toplam hacim için IRG-H2O 1.85 ml, 0.75 ml: bir 125 ml tarafında kolun şişesine aşağıdaki ekleyin. </li><li> De-gazı olarak, adım 2&#39;de açıklandığı 10 dakika süreyle çözeltisi. </li><li> Istifleme jel solüsyonu gaz alma edilirken, çözülmesi jel üst 1X çözme jel tamponu ile doyurulmuş 1-bütanol dökünüz. IRG-2 H, O. ile bir kez çözülmesi jeli üstünde durulama Suyu dökün ve kalan sıvı emmek için bir ayyaş ya Whatman filtre kağıdı kullanın. </li><li> Lütfen vakum gazından arındırılmış çözeltisinden 3 ml çıkarın ve% 10 APS ve TEMED 2 ul 9 ul ekleyin ve iyice karıştırın. </li><li> Hızla kısa ön jel tabakası üst kenarına yakın olduğu kadar kaset tarafı aşağı çalışmasına izin, kaset çözüm dökün. </li><li> , Jel kaset üst kısmına yerleştirilmiş bir temiz jel tarak eklemebunu tutturmak için tarak üzerinden kaset üzerinde bağlayıcı klip yerleştirin. Istifleme jel gece için 1 saat polimerize edelim. </li></ol> 4. Jel saklama <ol><li> Jel tamamen polimerize zaman, bağlayıcı klipleri çıkarın ve ısıl yapışmalı plastik poşet içinde kaset, ya da yeniden yapışmalı plastik torbaya yerleştirin. </li><li> Kese içine% 0,1 sodyum azid içerir 1X istifleme jel tamponu 20 ml dökün. </li><li> 5 saniye çantası mühür ve çanta hiçbir sızıntı olmadığından emin olmak ısıtın. </li><li> 4 ° C&#39;de jel depolamak </li></ol> 5. Numunelerin Hazırlanması <ol><li> Ince duvarlı mikrosantrifüj tüpü, protein numune 6.5 ul, bir 2x Novex Tris-Glisin SDS numune tamponu, 100 mM ditiyotretol (DTT) ve IRG-H 2 O 1 ul (gerekirse) 7.5 ul ekleyebilirsiniz 15 ul toplam hacim. </li><li> Vorteks ile örnek karıştırmak ve kısa bir süre için borunun alt tarafına içeriği getirmek için örnek santrifüjlenir. </li> &lt;li&gt; denatüre 70 ° C Örnek 10 dakika için. </li></ol> 6. Jeli çalıştıran <ol><li> Novex jel alet ayarlayın. </li><li> Iç haznesine Nupage antioksidan 500 ul, ve Tris-Glisin dış tamponu çalıştırma odasına 300 ml içeren Tris-Glisin tampon Çalıştırma 200 ml ilave edilir. </li><li> Bir jel yükleme pipet kullanarak kuyulara denatüre protein örneklerinin (15 ul) yükleyin. </li><li> 60-70 dakika 200 V jel çalıştırın. </li></ol> 7. Jel boyama <ol><li> Jel cihazdan Kaseti çıkarın ve jel bıçak ya da sert bir spatula kullanarak arayla iki plastik jel plakları meraklı epoksi mühür kırmak. Jel yırtmayın özen, plakalardan jeli dikkatle ayırın. </li><li> IRG-H ile jel nazik ajitasyon ile 2 O üç kere durulanır. </li><li> Nazik ajitasyon ile 1 saat boyunca Invitrogen SimplyBlue SafeStain ile jel leke. </li><li> Boyama çözümü kapalı dökün veIRG-2 H, O. ile jel destain </li></ol> 8. Kasetleri Temizleme <ol><li> Tekrar tekrar böylece kasetleri tüm epoksi kazıyarak ya da soyma metal bir spatula kullanın. </li><li> Bir deterjan solüsyonu ile plastik jel plakları her yıkayın ve distile su takip musluk suyu ile iyice durulayın. </li></ol> 9. Temsilcisi Sonuçlar Epoksi bir su geçirmez sızdırmazlık, (Şekil 1) oluşturacak, çünkü, kaset akrilamid çözeltisi içine döküldü ve bunların olduğunda sızıntı olmayacaktır. Buna ek olarak, jel kasetleri kolayca boyanması için jel almak için açılabilir ve epoksi onların devam yeniden kullanılmasına izin vermek kasetlerinin sıyrılır edilebilir. Şekil 2 de gösterildiği gibi, jel protein belirteçler ve rutin protein elektroforez için uygun örnek gruplarından net ayrım gösterir. <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="Şekil 1" /> Şekil 1.. Staminigel kasetleri mühürlemek için epoksi katyondur. epoksi ince bir tabaka montajı için önceden kasetleri birinin ucuna uygulanır. Epoksi vurgulamak için, bu görüntü yanlış renkli kırmızı oldu. <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="Şekil 2." /> Şekil 2. Bir SDS-Tris-glisin jel üzerinde, proteinlerin ayrılması tekrar kullanılabilir minigel kasetleri kullanılarak döküldü. Yukarıda tarif edildiği gibi SDS-PAGE uygulanmıştır. Aşağıdaki elektroforez jel üreticinin talimatlarına göre SimplyBlue SafeStain ile boyanmış minigel kaset, kaldırılır ve aktarılan beyaz ışık kullanılarak fotoğraflandı. Lane M SeeBlue Plus2 öncesi lekeli protein standartları içerir. Şeritli M proteinlerin moleküler ağırlıkları kilodalton (kDa) içinde jel sağ tarafında gösterildiği edilir. Diğer bir şerit glutation-S-transferaz (GST)-etiketli proteini ifade eden bakteriler bir temizlenir lizat proteinleri göstermektedir. Gösterildiği proteinleri g yıkanmış olanlardırGST-etiketli protein elüsyon önce lutathione boncuk.

<ol>
	<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Raymond, S., Weintraub, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide+gel+as+a+supporting+medium+for+zone+electrophoresis.">Acrylamide gel as a supporting medium for zone electrophoresis.</a> Science. 130, 711-711 (1959).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem. Biophys. Res. Commun. 28, 815-820 (1967).</li></ol>

Çıkar çatışması ilan etti.

Protein jel elektroforez en moleküler biyoloji laboratuvarları için vazgeçilmez bir yöntemdir. Piyasada bulunan minigels rahatlık yüksek düzeyde sağlamak, ama pahalıya mal olabilir. Burada ticari minigels popüler bir marka minigel kasetleri yeniden nasıl kullanılacağını göstermektedir. Bu yöntem jellerin hazır bir kaynağı sahip kolaylık korur, ancak ticari minigels maliyetinin bir kısmını. Bu prosedür önemli adımlarından tamamen sızmasını önlemek için unpolymerized akrilamid eklemeden ?...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Invitrogen Nupage Novex Minigel Kaset (1.0 mm) </td><td> Invitrogen </td><td> NC2010 </td><td> 2, 5, 10, 12, 15 ve 2B kuyu mevcuttur </td></tr><tr><td> Plastik Epoksi </td><td> Loctite&#39;a </td><td> 108771 </td><td></td></tr><tr><td> Elektrik Teyp </td><td> İskoç </td><td></td><td></td></tr><tr><td> 125 mL yan kol şişesi </td><td> Borcam </td><td> 5360 </td><td></td></tr><tr><td> 4X Tris-Base çözme jel tamponu </td><td></td><td></td><td> 1.5 M Tris-bazlı pH 8.7 </td></tr><tr><td> akrilamid </td><td> Fisher Scientific </td><td> 01065-500 </td><td></td></tr><tr><td> Bis-akrilamit </td><td> MP Biomedicals, Inc </td><td> 800173 </td><td></td></tr><tr><td> % 30.8 &#39;akrilamid çözüm </td><td></td><td></td><td> 30:0.8 akrilamid: bis-akrilamid IRG-2 H O </td></tr><tr><td> % 10 APS </td><td> Fisher Scientific </td><td> BP179-25 </td><td> 1 ml IRG-H 2 O da 0.1 g amonyum persülfat, kullanımdan hemen önce hazırlanan </td></tr><tr><td> TEMED </td><td> Arcos Organics </td><td> 138450500 </td><td></td></tr><tr><td> 1-bütanol 1X Ayrıştırıcı jel tamponu ile doyurulmuş </td><td></td><td></td><td> 1X çözme jel tampon 05:02 1-bütanol </td></tr><tr><td> 4X Tris-Base istifleme jel tamponu </td><td></td><td></td><td> 1.5 M Tris-bazlı pH 6.8 </td></tr><tr><td> Jel tarak </td><td> Invitrogen </td><td> NC3015 </td><td> 15 iyi, 1.0 mm kalınlık </td></tr><tr><td> Darbe Sealer </td><td> TEW Elektrikli Isıtma Ekipmanları </td><td></td><td></td></tr><tr><td> Isı Mühürlenebilir Kılıfı </td><td> Kapak / Scotchpak </td><td></td><td> 1 pint boyutu (6.5 &quot;x 8&quot;) </td></tr><tr><td> Jel depolama tamponu </td><td></td><td></td><td> 1X% 0,1 sodyum azid ile jel tamponu istifleme </td></tr><tr><td> Novex SDS Tris-Glisin numune tamponu (2X) </td><td> Invitrogen </td><td> LC2676 </td><td></td></tr><tr><td> DTT </td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X Tris-Gylcine Running tampon </td><td> Invitrogen </td><td> LC2675 </td><td> 10X stok 1L: 29.0 g Tris-Base, 144.0 g Glisin, 10,0 g SDS </td></tr><tr><td> XCell SureLock Mini-Hücre </td><td> Invitrogen </td><td> EI0001 </td><td></td></tr><tr><td> Jel Bıçak </td><td> Invitrogen </td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue Güvenli Leke </td><td> Invitrogen </td><td> LC6065 </td&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

Sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) analizi ile proteinlerin değerlendirilmesi biyokimya ve moleküler biyoloji araştırmacılar 1-4 tarafından sıkça kullanılan bir tekniktir. Proteinlerin günlük analizleri gerçekleştirmek için laboratuar, ticari olarak mevcut poliakrilamid jel (~ $ 10/gel) maliyetini önemli bir zaman içinde olabilir. Bu maliyeti azaltmak için, bazı araştırmacılar kendi poliakrilamid jelleri hazırlamak. Bu jellerin dökme Geleneksel yöntemler genellikle özel ekipman ve pahalı olması ve birçok jeller dökme ve gelecekteki kullanım için saklama engel olabilir cam jel plakları kullanmaktadır. Ayrıca, döküm temizleme veya jel sırasında cam plakalar ele lisans laboratuvar derslerinde kendi kullanılmasına engel olabilir, bir güvenlik tehlikesi yaratmak, kazaen kırılması neden olabilir. Bizim protokolü geri dönüşüm Invitrogen Nupage Novex minigel kasetleri aynı anda birden fazla protein jelleri dökün, ve ucuz ma gösteren nasılterials bir ev geliştirme mağazasından satın. Bu ekonomik ve akıcı yöntemi, birkaç hafta süreyle 4 ° C&#39;de jeller depolamak için bir yöntem içerir. Piyasada satılan jeller, sık sık protein jeller çalıştırmak laboratuarlarından plastik jel kasetleri yeniden kullanarak önemli maliyet tasarrufu ve çevre yardımcı olabilir. Ayrıca, plastik jel kasetleri lisans laboratuar sınıfları için ideal hale getiriyor kırılması, son derece dayanıklıdır.

Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Pouring and Running a Protein Gel by reusing Commercial Cassettes

Bizim protokolü bir ev geliştirme mağazasından satın dönüşüm Invitrogen Nupage Novex minigel kaset ve ucuz malzemeler ile aynı anda birden fazla protein jelleri dökün gösterilmiştir. Bu ekonomik ve akıcı yöntemi, birkaç hafta süreyle 4 ° C&#39;de jeller depolamak için bir yöntem içerir. Piyasada satılan jeller, sık sık protein jeller çalıştırmak laboratuarlarından plastik jel kasetleri yeniden kullanarak önemli maliyet tasarrufu ve çevre yardımcı olabilir.

Ticari kasetleri yeniden kullanılmasıyla Dökme ve Protein Jel Koşu

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

pouring-and-running-a-protein-gel-by-reusing-commercial-cassettes

Research

JoVE Journal

Biology

24.8K Görüntüleme.  University of Florida. Bizim protokolü bir ev geliştirme mağazasından satın dönüşüm Invitrogen Nupage Novex minigel kaset ve ucuz malzemeler ile aynı anda birden fazla protein jelleri dökün gösterilmiştir. Bu ekonomik ve akıcı yöntemi, birkaç hafta süreyle 4 ° C'de jeller depolamak için bir yöntem içerir. Piyasada satılan jeller, sık sık protein jeller çalıştırmak laboratuarlarından plastik jel kasetleri yeniden kullanarak önemli maliyet tasarrufu ve çevre y...

Video: Pouring and Running a Protein Gel by reusing Commercial Cassettes

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

Denatüre Gradient Jel Elektroforezi (DGGE)

Biyoloji

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Yarı denatüre Deterjan-Agaroz Jel Elektroforez Amiloid Toplama taraması

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

X-ray Kristalografi Protein Kristalizasyon

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Küçük Bispecific Affinity Tag Kolaylaştırılmış Ortogonal Protein Saflaştırma

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Rastgele Microseed Matrix ELEME Protein Belirginleşmesine Başarı Oranı artırma

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...