1. Подготовка образцов Первый шаг в западных промокательной является пробоподготовки. Для подготовки образцов для запуска геля клетки и ткани должны быть лизируются для освобождения белков, представляющих интерес. <ul><li> Удобный, готовый к использованию реагента, который является универсальным и простым в использовании для извлечения растворимого белка CytoBuster или PhosphoSafe. Эти добычи буферы содержат моющие средства оптимизированы для эффективного извлечения растворимых белков млекопитающих и клетки насекомых. Нежный, без ионного состава CytoBuster реагента извлечения белка позволяет изоляции функционально активных эндогенных или выразили белков без необходимости вторичной очистки, такие как ультразвуком или замораживания / оттаивания. PhoshoSafe имеет дополнительное преимущество, содержащий четыре ингибиторов фосфатазы. <ol><li> Гранул клеток центрифугированием низкой скорости (например, 5 мин при 2500 мкг), процедить осадок клеток хорошо. </li><li> Ресуспендируют клеток в CytoBuster белка Добыча реагента использовании 150 мкл на 10 6 клеток (оптимальное количество реагента CytoBuster извлечения белка может меняться в зависимости от размера ячейки). </li><li> Инкубируйте при комнатной температуре в течение 5 мин. </li><li> Трансфер в подходящую трубку и спина в течение 5 мин при 16 000 мкг при 4 ° C. </li><li> Передача очищается супернатант (клеточного экстракта) в новую пробирку и приступить к анализу. </li></ol></li><li> Для специализированных извлечения белка, мы рекомендуем использовать наши высококачественные ProteoExtract Kits. EMD имеет более десятка наборов для митохондриальных изоляции, субклеточном добычи протеома, добыче трансмембранного белка, и многие другие. Пожалуйста, посетите наши западные целевую страницу пятном для дальнейших подробностей. </li><li> EMD также продает широкий спектр протеаз и фосфатазы наборов ингибитор коктейль. Как только происходит лизис, протеолиз, дефосфорилирование, и денатурация начать. Эти события могут быть замедлены чрезвычайно если образцы хранятся на льду или при температуре 4 ° С во все времена и соответствующие ингибиторы добавляют в свежем виде лизис буфера. Пожалуйста, посетите наши западные целевую страницу пятном для дальнейших подробностей. </li></ul> 2. SDS-PAGE Второй шаг в западных промокательной является разделения белков. Белки отделить на основе молекулярной массой. <ul><li> Вы можете сделать свой собственный геля ПААГ, однако мы будем использовать коммерчески сборных геля. <ol><li> После подготовки образца, вы готовы для определения концентрации белка. EMD имеет два набора для этого, чтобы измерять концентрацию, один из которых без Вмешательство белка Kit пробирной, а другой Пробирной BCA белка Kit. </li><li> Как только концентрация белка оценивается мы готовы подготовить нашу выборку для загрузки геля. Антитела обычно признают небольшую часть белок (именуемые эпитопа), и этот домен может находиться внутри 3D конформации белка. Чтобы разрешить доступ антитела к этой части надо разворачиваться белка. </li><li> Для денатурации, используйте загрузки буфера с анионными денатурирующих моющего средства додецилсульфата натрия (SDS), и кипятить смесь на 95-100 ° С в течение 5 минут. Легкой и удобной погрузки буфер для использования 4X буфера проб. </li></ol></li><li> Поместите первую полосу хорошо Трейл маркерный белок Mix. Этот маркер имеет три ссылки полосы, которые позволяют контролировать электрофореза. Когда гель испачкается, 10 полос видны в пределах от 10-225 кДа. </li><li> Заполните электрофореза единицы проточной буфера. Для PAGE гелей, это, как правило 1X Трис-глицин. Для получения подробных рецептов буфера, посетить Западную промокательной странице. EMD предлагает высококачественные буферами реагентов с нашей линии химического OmniPur. Выполнить геля при 220 В в течение 1 часа. </li><li> Как только белки разделены, пятно геля Кумасси синий обеспечить белки мигрируют ровно и равномерно. RAPIDstain является сверхчувствительных пятно, которое готово к использованию. Нет destaining не требуется. </li></ul> 3. Белка-переносчика Так же, как белки с электрическим зарядом могут быть вызваны путешествовать по геля в электрическом поле, так что может быть передано белков в электрическом поле из геля на мембрану, быть или PVDF или нитроцеллюлозы. <ul><li> Сегодня мы будем делать мокрой передачи. В мокрых передачи, гель и мембрана зажаты между губкой и бумаги (губки / бумага / гель / мембрана / бумага / губка) и все зажаты плотно вместе после того, гарантируя, что ни воздушные пузыри образовались между гелем и мембраной. Сэндвич погружен в передаче буфера для которых электрические поля. Отрицательно заряженные белки путешествия к положительно заряженному электроду, но мембрана останавливает их, связывает их и не позволяет им продолжать далее. </li><li> Два типа мембран доступны: нитроцеллюлоза и PVDF. И работать хорошо. Сегодня мы будем использовать PVDF. PVDF мембраны требуют замачивания в метанол в течение нескольких минут, а затем ледяной буфера передачи в течение 5 минут. Гель также необходимо, чтобы уравновесить в течение нескольких минут в ледяной буфер передачи. Невыполнение этого требования может сократиться гель при передаче. </li><li> Буфера для влажной передачи 1X Трис-глицин, с 20% метанола. Подробная подготовка буфера доступны на нашем сайте. </li><li> После завершения передачи, это хорошая идея, чтобы визуализировать белки, чтобы гарантировать даже передачи без каких-либо воздушных карманов. Это можно легко сделать с Blot RedAlert Западной пятно. Это пятно является готовой к использованию и destaining можно легко сделать с водой. </li></ul> 4. Антитела и аксессуаров Реагенты <ul><li> Первым шагом в этом зондирования и антитела к антигену, чтобы блокировать мембраны. Блокировка мембрана предотвращает неспецифическую фоне обязательными. </li><li> Либо обезжиренного молока или БСА может быть использован как блокирование решения. Однако при использовании фосфорно-специфические антитела, это не рекомендуется использовать молоко, то это вызовет высокий фон. </li><li> EMD предлагает несколько высококачественных блокирование реагентов, в том числе SeaBlock, которая не является млекопитающим блокирующий агент и промокните QuickBlocker, который готовят, готовый к использованию блокирующий агент. У нас также есть высококачественные BSA (доля V) имеется. <ol><li> Инкубируйте мембраны 1 час при комнатной температуре, при легком волнении. </li><li> Вымойте три раза в TBST или PBST после инкубации. Легкий и удобный способ сделать TBST или PBST является использование нашего таблетки (PBS ТВИН Таблетки / TBS ТВИН таблетки) </li></ol></li><li> Как только мембрана была заблокирована, вы готовы для инкубации с первичными антителами. EMD года предлагает полный портфель исключительных антител с лучшими антител гарантии в промышленности на протяжении более 30 лет. Помните, что все антитела EMD, мы предлагаем полный 100% без риска гарантии. Покупка антитела и использовать его для любых видовой специфичности или приложения вы хотите. Если антитела не работает, чтобы Вы остались довольны, мы предоставим полный кредит, который будет использоваться на любой другой продукт. Для более подробной информации, пожалуйста, посетите наши западные промокательной веб-страницы. <ol><li> Сегодня мы будем инкубации нашей главной антител при решении 1 из SignalBoost. SignalBoost большой продукт, позволяющий вашему сигналу быть существенно увеличены. Просто инкубируйте ваших первичных антител в растворе 1 и инкубировать в течение 1 часа или, как вы это обычно делаете. Существует нет необходимости добавить дополнительные блокаторов. Кроме того, вы можете использовать BSA или обезжиренное сухое молоко, как блокирующий агент. </li><li> Промыть TBST. Мы делаем TBST использованием готовых к растворяются TBST таблетки. </li><li> Инкубируйте ваших вторичных антител при решении 2 из SignalBoost. Инкубируйте вторичные антитела в блокирующем буфере в течение 1 часа. Вымойте мембраны еще несколько раз с TBST. </li></ol></li></ul> 5. Обнаружение Для сопряжения HRP вторичными антителами, ECL традиционно используется. Отличные ECL реагента мы предлагаем RapidStep. Преимущества RapidStep в том, что нет смешивания просвета или усилитель не требуется. Просто распылите мембраны несколько раз, и развивать с помощью рентгеновской пленки. RapidStep предлагает низкую чувствительность пиктограмма, а также излучать свет в течение 2 часов после того субстрата.

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Renart, J., Reiser, J., Stark, G. R., R, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc Natl Acad Sci U S A. 76 (7), 3116-3120 (1979).</li><li>Burnette, W. N. '. W. e. s. t. e. r. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=blotting':+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate+polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li><li>Ambroz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+quantification+accuracy+for+Western+blots+Image+Analysis.">Improving quantification accuracy for Western blots Image Analysis.</a> , (2006).</li><li>Quan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species-mediated+endoplasmic+reticulum+stress+and+mitochondrial+dysfunction+contribute+to+cirsimartitin-induced+apoptosis+in+human+gallbladder+carcinoma+GBC-SD+cells.">Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to cirsimartitin-induced apoptosis in human gallbladder carcinoma GBC-SD cells.</a> Cancer Lett Article. , (2010).</li><li>O'Malley, D. e. r. v. i. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+colonic+corticotrophin-releasing+factor+receptors+in+the+maternally+separated+rat+model+of+irritable+bowel+syndrome:+Differential+effects+of+acute+psychological+and+physical+stressors.">Alterations in colonic corticotrophin-releasing factor receptors in the maternally separated rat model of irritable bowel syndrome: Differential effects of acute psychological and physical stressors.</a> Peptides. 31 (4), 662-670 (2010).</li></ol>

Авторы Анна и Джесси Эслами Лухан являются сотрудниками EMD химических Inc, которая производит реактивы и инструменты, используемые в настоящей статье.

В этом видео-презентации, мы выделили основные шаги в западных промокательной которые пробоподготовки, SDS-PAGE, мембранные блокировка / зондирования с антителами, и обнаружения. За каждый шаг процесса, надлежащего протоколы и соответствующие реагенты должны быть использованы для достижения высоких результатов.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CytoBuster&trade; Protein Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71009</td>
<td><a href="http://www.emdbiosciences.com/cytobuster">www.emdbiosciences.com/cytobuster</a> <a href="http://www.merck4biosciences.com/cytobuster">www.merck4biosciences.com/cytobuster</a></td>
</tr>
<tr>
<td>PhosphoSafe&trade; Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71296</td>
<td><a href="http://www.emdbiosciences.com/phosphosafe">www.emdbiosciences.com/phosphosafe</a> <a href="http://www.merck4biosciences.com/phosphosafe">www.merck4biosciences.com/phosphosafe</a></td>
</tr>
<tr>
<td>Non-Interfering Protein Assay&trade; Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>488250</td>
<td><a href="http://www.emdbiosciences.com/noninterfering">www.emdbiosciences.com/noninterfering</a> <a href="http://www.merck4biosciences.com/noninterfering">www.merck4biosciences.com/noninterfering</a></td>
</tr>
<tr>
<td>BCA Protein Assay Kit </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71285</td>
<td><a href="http://www.emdbiosciences.com/BCA">www.emdbiosciences.com/BCA</a> <a href="http://www.merck4biosciences.com/BCA">www.merck4biosciences.com/BCA</a></td>
</tr>
<tr>
<td>4X SDS Sample Buffer </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70607</td>
<td><a href="http://www.emdbiosciences.com/4XSDS">www.emdbiosciences.com/4XSDS</a> <a href="http://www.merck4biosciences.com/4XSDS">www.merck4biosciences.com/4XSDS</a></td>
</tr>
<tr>
<td>Trail Mix&trade; Protein Markers</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70980</td>
<td><a href="http://www.emdbiosciences.com/trailmix">www.emdbiosciences.com/trailmix</a> <a href="http://www.merck4biosciences.com/trailmix">www.merck4biosciences.com/trailmix</a></td>
</tr>
<tr>
<td>ProteoExtract&trade; Protein Extraction Kits</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/proteoextract">www.emdbiosciences.com/proteoextract</a> <a href="http://www.merck4biosciences.com/proteoextract">www.merck4biosciences.com/proteoextract</a></td>
</tr>
<tr>
<td>OmniPur&reg; Products</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/omnipur">www.emdbiosciences.com/omnipur</a></td>
</tr>
<tr>
<td>RAPIDstain&trade; Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>553215</td>
<td><a href="http://www.emdbiosciences.com/rapidstain">www.emdbiosciences.com/rapidstain</a> <a href="http://www.merck4biosciences.com/rapidstain">www.merck4biosciences.com/rapidstain</a></td>
</tr>
<tr>
<td>RedAlert&trade; 10X Western Blot Stain</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71078</td>
<td><a href="http://www.emdbiosciences.com/redalert">www.emdbiosciences.com/redalert</a> <a href="http://www.merck4biosciences.com/redalert">www.merck4biosciences.com/redalert</a></td>
</tr>
<tr>
<td>SeaBlock&trade; Reagent, Salmon Plasma</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>558300</td>
<td><a href="http://www.emdbiosciences.com/seablock">www.emdbiosciences.com/seablock</a> <a href="http://www.merck4biosciences.com/seablock">www.merck4biosciences.com/seablock</a></td>
</tr>
<tr>
<td>BLOT-QuickBlocker Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>WB57</td>
<td><a href="http://www.emdbiosciences.com/quickblocker">www.emdbiosciences.com/quickblocker</a> <a href="http://www.merck4biosciences.com/quickblocker">www.merck4biosciences.com/quickblocker</a></td>
</tr>
<tr>
<td>Albumin, Bovine Serum, Fraction V</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>12660</td>
<td><a href="http://www.emdbiosciences.com/bovineserum">www.emdbiosciences.com/bovineserum</a> <a href="http://www.merck4biosciences.com/bovineserum">www.merck4biosciences.com/bovineserum</a></td>
</tr>
<tr>
<td>PBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524653</td>
<td><a href="http://www.emdbiosciences.com/PBST">www.emdbiosciences.com/PBST</a> <a href="http://www.merck4biosciences.com/PBST">www.merck4biosciences.com/PBST</a></td>
</tr>
<tr>
<td>TBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524753</td>
<td><a href="http://www.emdbiosciences.com/TBST">www.emdbiosciences.com/TBST</a> <a href="http://www.merck4biosciences.com/TBST">www.merck4biosciences.com/TBST</a></td>
</tr>
<tr>
<td>SignalBoost&trade; Immunoreaction Enhancer Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>407207</td>
<td><a href="http://www.emdbiosciences.com/signalboost">www.emdbiosciences.com/signalboost</a> <a href="http://www.merck4biosciences.com/signalboost">www.merck4biosciences.com/signalboost</a></td>
</tr>
<tr>
<td>RapidStep&trade; ECL Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>345818</td>
<td><a href="http://www.emdbiosciences.com/rapidstep">www.emdbiosciences.com/rapidstep</a> <a href="http://www.merck4biosciences.com/rapidstep">www.merck4biosciences.com/rapidstep</a></td>
</tr>		</tbody>
		</table>
		</div>

western blotting: sample preparation to detection

Вестерн-блоттинга является аналитический метод используется для обнаружения специфических белков в данном образце гомогената ткани или экстракта. Он использует гель-электрофореза для разделения нативных или денатурированных белков длина полипептид (денатурирующих условиях) или 3-D структуры белка (родной / не денатурирующих условиях). Белки затем переведен в мембране (обычно нитроцеллюлозы или PVDF), где их опроса (обнаружен), используя антитела, специфичные к белку-мишени.

Watch this Scientific Journal Video about Western Blotting: Sample Preparation to Detection at JoVE.com

Western Blotting: Sample Preparation to Detection

Вестерн-блоттинга является аналитический метод используется для обнаружения специфических белков в данном образце гомогената ткани или экстракта.

Вестерн-блоттинг: Подготовка образцов для обнаружения

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

western-blotting-sample-preparation-to-detection

Research

JoVE Journal

Biology

139.0K Views.  EMD Chemicals Inc.. Вестерн-блоттинга является аналитический метод используется для обнаружения специфических белков в данном образце гомогената ткани или экстракта.

Video: Western Blotting: Sample Preparation to Detection

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI пробоподготовки: ультра тонкий слой Метод

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

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

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

Вестерн-блоттинг Использование Invitrogen NuPage Novex бис Трис MiniGels

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

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Протеомный Подготовка образцов из фиксированных формалином и залитых парафином ткани

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

V3 Без пятен рабочий процесс для практического, удобного и надежного тотального контроля загрузки белка в Западном Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

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

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

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

Самый быстрый Западная в городе: современный поворот на анализ Классический вестерн-блоттинга

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

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Руководство по современной количественной Люминесцентная Вестерн-блоттинга с устранения неисправностей стратегий

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Муцин Электрофорез в агарозном геле: Вестерн блот для высокой молекулярной массой гликопротеинов


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Подготовка проб для анализа Масс-цитометрии

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Подготовка образцов и изображений Exosomes, просвечивающей электронной микроскопии

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Миниатюрных Пробоподготовка для просвечивающей электронной микроскопии

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...