Dr. Nicola Wiethölter Biz 1-D jellerin hazırlanması ve boyama için teşekkür ederiz. Bu çalışma Deutsche Forschungsgemeinschaft (ND ve SM GRK 1089/project 5) hibe tarafından finanse edilen ve ND için Stiftung Jürgen Manchot bir araştırma bursu ile desteklenmiştir.

Bölüm 1: fincan yükleme kullanarak, iki boyutlu (2-D) jel elektroforezi <ol><li> IPG şerit rehidrasyon ve izoelektrik odaklama (IEF) <ul><li> 125 ul rehidratasyon çözüm Immobiline DryStrip jeller, pH 6-11 rehidrate (7 cm) [7 M üre, 2 M tiyoüre,% 4 ahbap, 50 mM hydroxyethyldisulfide IPG Tampon pH 6-11 ve% 2] Immobiline DryStrip Reswelling Tepsi en azından 10 saat. </li><li> IEF örnek tampon çöktürülmüş protein örnek çözülür [7 M üre, 2 M tiyoüre,% 2 ahbap, Tampon pH 6-11 IPG ASB-14, 50 mM hydroxyethyldisulfide ve% 2], 100 ul başına 60-100 mg protein karşılık gelen% 2 . </li><li> Çözünürleştirme (oda sıcaklığında en az 30 dakika) sonra, numune kaplarını kullanarak fincan yükleme anodik ile protein örnek geçerlidir. Fincan hacmi 100 ul (Manifold bardak ul 150 kadar izin verin). Not: hala fincan sıvı bir film varken odaklama protokol ve dolum bardak başında düşük voltajlı bir adım eklerseniz büyük örnek miktarda yükleyebilirsiniz ! </li><li> Multiphor II Elektroforez Ünitesi degrade modunda 11.1 KVH için izoelektrik odaklama gerçekleştirin. </li></ul></li><li> Denge ve SDS-PAGE <ul><li> IEF sonra, IPG şeritler, çalkalayıcı dengeleme çözüm sırasıyla% 1 ve% 2.5 dithiothreitol iodoacetamide kullanarak her seferinde 15 dakika azaltılması ve alkilleme tabi tutulmuştur [50 mM Tris-HCl/pH 8.8, 6 M üre,% 30 gliserol ve% 2 SDS]. </li><li> H 2 O ile dengelenmiş şeritler durulayın ve aşırı dengeleme tampon kaldırmak için bunları Vatmanlar kağıda kurulayın. Daha sonra SDS çalışan tampon (1X) şeritler daldırma. </li><li> İkinci boyut toplam akrilamid konsantrasyonu% 12 ile dikey elektroforez sistemleri SDS-PAGE ile yapılır. Dengelenmiş IPG şeritleri ayıran jeller üstüne yerleştirilir ve sıcak agaroz çözüm [Bromophenol Mavi içeren tampon çalıştıran% 0.5 agaroz] ile fikse edildi. </li><li> Proteinler, 120 V tarafından takip boya ön jel kaset altına ulaşana kadar 15 dakika için 80 V ile başlayan, 2.5 saat için ayrıldı. </li></ul></li></ol> Bölüm 2: G-250 ile CBB Kolloidal Coomassie boyama 1. Boyama çözümler Not: Millipore sistemleri (Milli-Q su), analitik dereceli (pa) ve yüksek saflıkta su gibi yüksek kalite ile boyama çözümler hazırlanması için kimyasal maddeler kullanmayın. <table border="0"><tbody><tr><td colspan="2"> Coomassie çözüm: </td><td> reklam 2000 ml H 2 O </td></tr><tr><td> % 0,02 (w / v) </td><td> CBB G-250 </td><td> 0,4 g </td></tr><tr><td> % 5 (w / v) </td><td> -hidrat alüminyum sülfat (14-18) </td><td> 100 g </td></tr><tr><td> % 10 (v / v) </td><td> etanol (% 96) </td><td> 200 ml </td></tr><tr><td> % 2 (v / v) </td><td> ortofosforik asit (% 85) </td><td> 47 ml </td></tr></tbody></table> Not: boyama çözümün hazırlanması için, aşağıdaki sıraya göre sıralı ek bileşenlerin muhafaza edilmelidir: <ol><li> Milli-Q su alüminyum sülfat çözülür </li><li> bundan sonra, etanol ekleyip homojenize ve çözüm CBB G-250 karışımı </li><li> çözüm tamamen eriyene kadar son zamanlarda, fosforik asit (asit dahil alkollü medya onların kolloidal haline agrega Coomassie moleküller sağlar) </li><li> Sonunda Milli-Q su ile doldurmak </li></ol> Not: son boyama çözüm koyu yeşil-mavi bir görünüme sahiptir ve parçacıkların etrafında yüzme doludur: <ul><li> Süzüntü bu çözüm değil! </li><li> Hazırlık amacıyla değiştirdiyseniz, daha az duyarlı bir daha mor-mavi bir çözüm olacaktı. </li></ul><table border="0"><tbody><tr><td colspan="2"> Çözüm Destaining: </td><td> reklam 2000 ml H 2 O </td></tr><tr><td> % 10 (v / v) </td><td> etanol (% 96) </td><td> 200 ml </td></tr><tr><td> % 2 (v / v) </td><td> ortofosforik asit (% 85) </td><td> 47 ml </td></tr></tbody></table> 2. Prosedürü Boyama <ul><li> İkinci boyut ayrıldıktan sonra, cam plakalar dikkatle jeller kaldırmak ve Milli-Q su ile dolu bir boyama çanak içine aktarabilirsiniz. </li><li> Milli-Q su ile jeller üç kere yıkayın yatay bir çalkalayıcı (yetersiz yıkama jelleri kalan SDS, protein, boya sınırlayıcı rahatsız edici olduğundan kötü hassasiyet neden) 10 dakika. </li><li> Coomassie çözüm kolloidal parçacıklar eşit dağıtmak için kullanmadan önce iyice çalkalayın. </li><li> 2-12 saat çalkalayıcı üzerinde ajitasyon Coomassie çözümü ile kaplı jeller inkübe edin. Bu boyama arasında boyama ilerlemeyi gözlemlemek, böylece marjinal arka plan üretir. Not: 10 dakika sonra ilk protein lekeleri inkübasyon 2 saat içinde yaklaşık% 80, bu boyama görünen görebilirsinizs maksimum düzeyde tamamlandı. Yaklaşık% 100 boyama için, gecelik inkübasyon önerilir. Bazı durumlarda, lekeli protein miktarı çok büyük ve parlak mavi çözüm dönüşür, boyama çözümün bir yenileme gerekli. </li><li> Boyama işleminden sonra Coomassie çözüm kaldırmak ve jeller iki kez Milli-Q su ile durulayın. Not: parçacıkları hala kalan boyama çözüm sürece yeniden kullanabilirsiniz, aksi takdirde (laboratuvar atık bertarafı için özel düzenlemeler olabileceğini akılda tutmak) atın. </li><li> Boya taneciklerini yapıştırarak, tüy bırakmayan bir kağıt havlu ile leke tabağına çıkartın ve destain 10 - 60 dakika. Not: Ayrıca jeller sadece Milli-Q su ile yıkayabilirsiniz ama tarama işlemi sırasında ortaya çıkacak jeller üzerinde düzensiz, zayıf Coomassie film sürecek, çünkü bu bizim için sadece hazırlık jeller tavsiye . </li><li> Son olarak Milli-Q su ile iki kez jeller durulama: jeller orijinal kalınlığına ulaşmak (alkol-asit ortam jeller daralma yapar) ve su nötralizasyon ek Coomassie renk yoğunluğunu artırır. </li></ul> Bölüm 3: Temsilci sonuçları Yukarıda açıklanan protokol takip ederseniz, 2-D jel ayırt edici giderilmiş ve iyi lekeli koyu mavi protein noktalar olsun. Hatta Shevchenko ve arkadaşları 5, kütle spektrometresi ile iyi bir duyarlılık ve uyumluluk iddia eden bir protokoldür göre gümüş lekeli bir 2-D jel göre, biz eşit boyama sonuçları elde etmek . <img src="/files/ftp_upload/1431/fig1.jpg" alt="Şekil 1" /> Şekil 1: Yukarıda açıklanan deney Nihai sonuç. Kang&#39;ın Coomassie protokolü (A), Shevchenko ve arkadaşları göre gümüş boyama gibi güçlü bir şekilde gerçekleştirir. (B) 2-D jel elektroforezi sonra proteinleri tespit . Bölüm 4: İpuçları ve püf noktaları <ul><li> Kendi örnekleri işlemeden önce bazı test renklendirmeler gerçekleştirin. Açıklanamaz nedenlerden dolayı bir ilgi protein başarıyla miligram uygulandı bile tespit edilebilir olabilir bazen bildirdi. Bu durumda biz duyarlılık test etmek için bazı seyreltme bir dizi hazırlamak için önerilir. </li><li> SDS-PAGE sırasında ayıran jel istifleme genel başarısız transfer protein jel algılama (ler) düşük varsa muhtemelen sebep olacaktır. Istifleme alanı da dahil olmak üzere tam jel boyama gibi yapışkan proteinlerin kontrol edebilirsiniz. </li><li> Izoelektrik odaklama sonuçları hızlı bir doğrulama için, doğrudan başka bir ikinci boyut ayırımı olmadan IPG-şeritler leke yapabilir. </li><li> (Jeller karşı kayıtsız devam ederseniz, boya jel bir tarafa bağlanır), her iki taraftan eşit şekilde boyanmış böylece Bazen boyama işlemi sırasında jeller açmak. </li><li> Biz karanlık bir şişe boyama çözüm (4 aya kadar şeffaf şişelerde) depolama ömrünü artırır yaşadı. </li><li> Boyama ve belgeleyen (asidik eklerseniz mantarlar tarafından Bulaşmayı önlemek) jeller birkaç yıl boyunca buzdolabında tutabilirsiniz sonra. </li><li> Triptik sindirmek için jel fişlerinin Destaining bir blok ısıtıcı ısınmanın hızlandırılabilir. </li></ul>

<ol>
	<li>Fazekas de St Groth, S., Webster, S. R. G., Datyner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+new+staining+procedures+for+quantitative+estimation+of+proteins+on+electrophoretic+strips.">Two new staining procedures for quantitative estimation of proteins on electrophoretic strips.</a> Biochim. Biophys. Acta. 71, 377-391 (1963).</li><li>Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+staining+of+proteins+in+polyacrylamide+gels+including+isoelectric+focusing+gels+with+clear+background+at+nanogram+sensitivity+using+Coomassie+Brilliant+Blue+G-250+and+R-250.">Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.</a> Electrophoresis. 9, 255-262 (1988).</li><li>Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., Orecchia, P., Zardi, L., Rigetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+Silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue Silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25, 1327-1333 (2004).</li><li>Kang, D., Gho, S. G., Suh, M., Kang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+and+Fast+Protein+Detection+with+Coomassie+Brilliant+Blue+in+Sodium+Dodecyl+Sulfate-Polyacrylamide+Gel+Electrophoresis.">Highly Sensitive and Fast Protein Detection with Coomassie Brilliant Blue in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.</a> Bull. Korean Chem. Soc. 11, 1511-1512 (2002).</li><li>Shevchenko, A., Wilm, M., Vorm, O., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+sequencing+of+proteins+silver-stained+polyacrylamide+gels.">Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.</a> Anal. Chem. 68, 850-858 (1996).</li></ol>

Yenilikçi ya da sadece başka bir Coomassie protokol? Şu anda Coomassie Brillant Blue prosedürleri boyama için birden fazla protokol var. Çoğu Neuhoff ve meslektaşları 2 tarafından en yaygın olarak kullanılan protokoller küçük veya büyük değişiklikler sonucu. Ayrıca Kang&#39;ın protokolü Neuhoff formülü dayanmaktadır. Ama gerçekten proteomik araştırma için alternatif Coomassie boyama yöntemi nedir? Biz diğer Coomassie protokolleri ve hatta gümüş ve...

<table border="1" ><tr><td> Ürün Adı </td><td> Şirket </td><td> Katalog No. </td><td> Yorumlar </td></tr><tr><td> : IEF ve SDS-PAGE </td><td></td><td></td><td></td></tr><tr><td> Immobiline DryStrip jeller </td><td> GE Healthcare </td><td> 17-6001-94 </td><td> Tarih sona 2 yıl sonra bile kullanılabilir. </td></tr><tr><td> Immobiline DryStrip Reswelling Tepsi </td><td> GE Healthcare </td><td> 80-6371-84 </td><td> Organik çözücüler ile temizlemeyin. 7-18 cm IPG-şeritler için tasarlanmıştır. </td></tr><tr><td> Immobiline DryStrip Kit </td><td> GE Healthcare </td><td> 18-1004-30 </td><td> Bir tepsi, elektrot sahibi, anot ve katot elektrotları, hizalama ve numune kabı bar ve numune kaplarını içerir. </td></tr><tr><td> EPS 3501 XL Güç Kaynağı </td><td> GE Healthcare </td><td> 18-1130-05 </td><td> Malzemeleri 3500 kadar gerilimi V </td></tr><tr><td> Multiphor II Elektroforez Ünitesi </td><td> GE Healthcare </td><td> 18-1018-06 </td><td> Hareketli elektrotlar tüm uzunluğu IPG şeritler halinde IEF etkinleştirmek (7-24 cm şeritler IPG) </td></tr><tr><td> PerfectBlue jel sistemi Twin S </td><td> Peqlab </td><td> 45-1010-C </td><td> 10x10 cm mini-jel formatında SDS-PAGE. Jel odasının bir soğutma sistemi içerir. </td></tr><tr><td> : Kolloidal Coomassie G-250 boyama için </td><td></td><td></td><td></td></tr><tr><td> kapakları ile yemekleri boyama </td><td> VWR </td><td> 216-3412 </td><td> Mini jeller için uyar. Çalkalamalı İstiflenebilir. </td></tr><tr><td> alüminyum sülfat-18-hidrat </td><td> Merck </td><td> 1.01102.5000 </td><td> Merck ile en iyi deneyimi yaptı. Sadece 5 kg&#39;lık paket kullanılabilir. </td></tr><tr><td> ortofosforik asit </td><td> Prolabo </td><td> 20 624,295 </td><td> Cam şişelerde satılır. </td></tr></table>

fast and sensitive colloidal coomassie g-250 staining for proteins in polyacrylamide gels

Coomassie Brillant Blue (CBB), genellikle basit bir boyama prosedürü ve yüksek kantitatif sunan SDS-PAGE ile ayrılmış proteinlerin görselleştirme için kullanılan bir boya. Ayrıca, kitle spektrometrik protein kimlik ile tamamen uyumlu. Ancak bu avantajlara rağmen, CBB, gümüş ya da floresan renklendirmeler daha az duyarlı ve bu nedenle analitik jel tabanlı proteomik yaklaşımlar proteinlerin tespiti için kullanılan nadir olarak kabul edilir. Orijinal Coomassie protokolü 1 CBB duyarlılığını artırmak için bazı geliştirmeler yapılmış. Kolloidal parçacıkları içine boya molekülleri dönüştürerek düşük-bol proteinlerin tespiti geliştirmek için iki önemli değişiklikler getirilmiştir: 1988 yılında, Neuhoff ve arkadaşları CBB G-250 tabanlı boyama çözüm 2,% 20 metanol ve uygulanan yüksek konsantrasyonlarda amonyum sülfat 2004 Candiano ve ark. Mavi Gümüş, fosforik asit, amonyum sülfat ve metanol 3 varlığı ile G-250 CBB kullanarak kuruldu . Bununla birlikte, tüm bu değişiklikler sadece yaklaşık 10 ng protein bir algılama sağlar. Kolloidal Coomassie boyama için yaygın fameless bir protokol Kang ve ark tarafından 2002 yılında yayımlandı. Kompleks maddeler ile ilgili Neuhoff kolloidal CBB boyama protokol değiştirilmiş. Bunun yerine amonyum sülfat, alüminyum sülfat ve metanol yerine daha az toksik etanol 4 kullandı . Roman alüminyum bazlı Kang&#39;ın çalışmada boyama biraz hassasiyet varyasyon proteinleri bağlı olarak 1 ng / bant (fosforilaz b) gibi düşük algılar üstün hassasiyet gösterdi. Burada, hızlı ve hassas analitik amaçlarla proteinlerin kolloidal Coomassie boyama Kang&#39;ın protokolü uygulaması göstermektedir. Biz rutin çalışma grubunda yapılan iki boyutlu jeller kullanarak hızlı ve kolay bir protokol gösterecektir.

Watch this Scientific Journal Video about Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels at JoVE.com

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Bu video Kang göre kolloidal Coomassie G-250 boyama protokol sevdirmek olacaktır<em&gt; Ve ark.</em&gt; Jeller ortalama 4 ng protein tespiti için. Boyama ve herhangi bir çaba olmadan 2 saat içinde tamamlanır. Biz rutin analitik amaçlar için jel bazlı proteomik Kang&#39;ın protokolü kullanın.

Proteinler için Poliakrilamid Jeller Hızlı ve Hassas Kolloidal Coomassie G-250 Boyama

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

fast-sensitive-colloidal-coomassie-g-250-staining-for-proteins

Research

JoVE Journal

Biology

40.0K Görüntüleme.  University Duesseldorf. Bu video Kang göre kolloidal Coomassie G-250 boyama protokol sevdirmek olacaktır Ve ark. Jeller ortalama 4 ng protein tespiti için. Boyama ve herhangi bir çaba olmadan 2 saat içinde tamamlanır. Biz rutin analitik amaçlar için jel bazlı proteomik Kang'ın protokolü kullanın.

Video: Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Jeller içinde Proteinler Boyama

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Biyoloji

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Chick Tüm Dağı Antikor Boyama Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denatüre Üre Poliakrilamid Jel Elektroforez (Üre SAYFA)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Hızlı Kalsiyum Dekapanları kardiyomiyositlerde Ölçme

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Hassas, Büyük Ölçekli Kantitatif metabolomik için bir strateji

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

Deniz Memelileri Miyoglobin Tespiti için bir Kolloidal Altın merkezli İmmunokromatografik Testi Strip Gelişimi

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Hassas immünohistokimyasal sitozin Modifiye Formlar Algılama

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

İnsan Beyin Dokusundan Proteinlerin Çoklu Reaksiyon İzleme Tabanlı Tespiti Kullanılarak Nicel Proteomik İş Akışı

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

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 ...