1。样品制备在免疫印迹的第一步是样品制备。为了准备运行的一种凝胶体细胞和组织样本需要，裂解释放的利益蛋白质。 <ul><li>一个方便，随时可以使用的试剂，这是通用和易于使用的可溶性蛋白提取CytoBuster或PhosphoSafe。这些提取缓冲区包含从哺乳动物和昆虫细胞中高效提取可溶性蛋白优化的洗涤剂。温柔，非离子组成CytoBuster蛋白抽提试剂，使隔离功能活跃的内源性或表达的蛋白质，而不需要为二级处理，如超声或冻结/解冻。 PhoshoSafe包含四个磷酸酶抑制剂的额外的好处。 <ol><li>颗粒细胞低速离心（如5分钟在2500 XG），漏细胞沉淀。 </li><li>重悬在CytoBuster蛋白抽提试剂使用150μL（CytoBuster蛋白抽提试剂细胞大小的最佳数量可能会有所不同），每10 6细胞的细胞。 </li><li>在室温下孵育5分钟。 </li><li>转移到一个合适的管和自旋为16000 XG 5分钟，在4 ° C。 </li><li>转让清除上清液（细胞提取物）到一个新的管，并进行分析。 </li></ol></li><li>专门蛋白提取，我们建议使用我们的高品质的ProteoExtract工具箱。 EMD有十几家线粒体隔离，亚细胞蛋白质组的提取，跨膜蛋白的提取，以及许多其他的套件。进一步详情，请访问我们的免疫印迹登陆页面。 </li><li> EMD还销售多种蛋白酶和磷酸酶抑制剂的鸡尾酒集。只要发生裂解，水解，去磷酸化和变性的开始。这些事件可以大大减慢，如果样品是在冰上或保持在4 ° C是在任何时候和适当的抑制剂增添了新鲜的裂解液。进一步详情，请访问我们的免疫印迹登陆页面。 </li></ul> 2。 SDS - PAGE电泳在西方印迹的第二步是蛋白质的分离。根据分子量蛋白质分离。 <ul><li>您可以使自己的网页凝胶，但是我们将使用一个商业预制凝胶。 <ol><li>准备您的样品后，您已经准备好，以确定蛋白质浓度。 EMD还有两个包来衡量浓度，一个是无干扰的蛋白质检测试剂盒和其他BCA蛋白检测试剂盒。 </li><li>蛋白浓度是评估后，我们随时准备为我们的样本中加载凝胶。抗体通常认识到一个感兴趣的蛋白质的一小部分（简称表面抗原），此域名可能驻留在蛋白质的三维构象。为了使这部分有必要开展的蛋白质抗体的访问。 </li><li>变性，变性的阴离子洗涤剂十二烷基硫酸钠（SDS）使用一个样缓冲液，煮沸5分钟的混合物在95-100 ° C。使用一种简单，方便装载缓冲4X采样缓冲器。 </li></ol></li><li>道上混的蛋白Marker负载以及第一线。这标志有三个参考乐队，让你可以监控电泳。当凝胶染色，10波段可见范围从10-225 kDa的。 </li><li>填充运行缓冲液的电泳单位。对于PAGE凝胶，这是通常1X Tris -甘氨酸。如需详细缓冲食谱，请访问Western印迹页。电解二氧化锰与我们的OmniPur化工行提供高品质的缓冲液试剂。 1小时，运行在220V的凝胶。 </li><li>一旦分离出来的蛋白质，考马斯蓝染色的凝胶，以确保均匀，均匀的蛋白质已迁移。 RAPIDstain是超敏感的污点，这是准备使用的。没有脱色是必需的。 </li></ul> 3。蛋白转移正如电荷的蛋白质可诱发穿行在一个电场凝胶，因此可以将蛋白质转移到一个电场，从凝胶上膜，PVDF或硝酸纤维素。 <ul><li>今天，我们将尽湿的转移。在调湿，凝胶和膜夹在海绵和纸（海绵/纸/凝胶/膜/纸/海绵），都紧紧夹住后，确保无气泡，凝胶和膜之间形成。三明治是淹没在电场应用到传输缓冲区。旅游对带正电荷的电极的，负电荷的蛋白质，但膜停止它们，它们绑定，并阻止他们继续进行。 </li><li>两种类型的膜：硝酸纤维素和聚偏氟乙烯。以及它们的工作原理。今天我们将使用聚偏氟乙烯。 PVDF膜需要浸泡在methano升了几分钟，冰冷转印缓冲液5分钟。凝胶也需要平衡在冰冷的传输缓冲区几分钟。如果不这样做，可能会在传输过程中的收缩凝胶。 </li><li>调湿的缓冲区是用20％甲醇1X Tris -甘氨酸。我们的网站上提供详细的缓冲准备工作。 </li><li>一旦传输完成，这是一个好主意，可视化的蛋白质，以确保没有气泡，甚至转移。这可以很容易地与RedAlert免疫印迹染色。这个污点是准备使用的，可以很容易与水脱色。 </li></ul> 4。抗体及辅助试剂<ul><li>在做探测和抗体的抗原的第一步，是块膜。阻断膜，防止非特异性背景约束力。 </li><li>要么不低脂牛奶或BSA可以被用来作为一个阻塞的解决方案。然而，当使用磷酸化特异性抗体，这是不建议使用牛奶，因为它会导致高背景。 </li><li>电解二氧化锰提供几个高品质的阻塞包括SeaBlock试剂，这是一个非哺乳动物的阻断剂的印迹QuickBlocker，这是准备，随时可以使用的阻断剂。我们也有高品质的BSA（组分V）提供。 <ol><li>在室温下轻轻摇动下孵育膜1小时。 </li><li>孵化后在TBST或PBST清洗三次。一个简单方便的方式，使TBST或PBST使用我们的片（PBS吐温片/ TBS电视台吐温片） </li></ol></li><li>一旦膜已被封锁，你准备的主要抗体孵育。电解二氧化锰已在行业中最好的抗体超过30年的保证提供全面的产品组合的特殊抗体。请记住，所有EMD的抗体，我们提供了一个完整的100％保证无风险。购买一种抗体，它使用任何物种特异性或应用程序，你的愿望。如果抗体不工作，您的满意，我们将提供一个完整的信用卡被用于任何其他产品。有关详细信息，请访问我们的免疫印迹网页。 <ol><li>今天，我们将培育我们的主要抗体，使用的SignalBoost解决方案1。 SignalBoost是一个伟大的产品，让你的信号大大加强。简单的解决方案1孵化您的主要抗体，孵育1小时，或像往常一样。有没有需要进一步补充阻断剂。或者，您可以使用BSA或脱脂奶粉作为阻断剂。 </li><li>洗涤用TBST。我们TBST使用现成的TBST溶解平板。 </li><li>孵育二级抗体使用的SignalBoost解决方案2。孵育二级抗体阻断1小时的缓冲。再次洗膜用TBST几次。 </li></ol></li></ul> 5。检测辣根过氧化物酶偶联的二抗，ECL是传统上用来。我们提供一个很好的ECL试剂RapidStep。 RapidStep的优点是没有管腔或增强的混合要求。只需喷膜几次和开发利用X射线胶片。 RapidStep提供的基板此外后​​象形图的灵敏度低，以及长达2个小时的发光。

<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>

作者安娜Eslami和杰西卢汉受雇于EMD化学公司生产的试剂和仪器在本条中使用。

在这个视频演示，我们已经强调了免疫印迹的样品制备的主要步骤，SDS - PAGE电泳，膜抗体阻断/探测，和检测。对于过程的​​每一个一步，适当的协议和相应的试剂应使用，以达到高品质的结果。

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		<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

Western印迹分析技术，用于检测特定的蛋白质，在一个给定的样本，组织匀浆或提取。它采用凝胶电泳分离长度的多肽（变性条件下），或由本地或变性蛋白的蛋白质三维结构（本机/非变性条件）。然后转移到一个膜（通常是硝酸纤维素或PVDF），他们在那里探测（检测）使用特定的靶蛋白的抗体的蛋白质。

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

Western Blotting: Sample Preparation to Detection

Western印迹分析技术，用于检测特定的蛋白质，在一个给定的样本，组织匀浆或提取。

免疫印迹检测样品制备

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

138.9K 次观看.  EMD Chemicals Inc.. Western印迹分析技术，用于检测特定的蛋白质，在一个给定的样本，组织匀浆或提取。

视频: 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样品制备：超薄层的方法

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

科研

生物学

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.

免疫印迹使用Invitrogen公司NuPage NOVEX双三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.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

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 无污渍工作流，适用于西式印迹中实用、方便和可靠的总蛋白质加载控制

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.

最快在西镇：对经典蛋白质印迹分析现代风味

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.

指南现代荧光定量免疫印迹与故障排除策略

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.

粘蛋白琼脂糖凝胶电泳：蛋白质印迹高分子量糖蛋白


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.

样品制备质谱术分析

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.

透射电镜下体的样品制备与成像

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.

透射电镜的小型化样品制备方法

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