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

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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

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 ...

Biyoloji

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 ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Glikozamid Jel Elektroforez ve Gümüş Boyama ile Glikozaminolikanların Tespiti

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Farelerde Progresif Direnç Eğitimi için Basit ve Ucuz Bir Koşu Tekerleği Modeli

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez Çalışmalarının Basitleştirilmesi

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Yazar Spot Işığı: Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez ve Damar Geçirgenliğinin Araştırılması

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Anjiyogenez Çalışmaları için Modifiye In Vivo Matrix Jel Plug Testi

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...