Name: western blotting using the invitrogen nupage novex bis tris minigels
Uploaded: 2007-08-22T00:00:00
Description: Watch this Scientific Journal Video about Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels at JoVE.com

这项工作是由来自美国国立卫生和乔治E ·休伊特基金会奖学金（美联社）研究院的拨款支持。

凝胶电泳 技术说明 ：在启动程序之前，您的蛋白质样品准备。 在这第一步，样品中的蛋白质，根据其分子量使用变性聚丙烯酰胺凝胶电泳（PAGE）分离。锂十二烷基硫酸钠（LDS）中加载NuPAGE ® LDS样品缓冲液保持在变性状态，多肽，蛋白质样品已在70 ° C加热10分钟一次。强还原剂配合使用，以消除二级和三级结构（数?...

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	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+Migration+chart:+.">Gel Migration chart: .</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+western-blot+protocol+from+the+Invitrogen+website.">Complete western-blot protocol from the Invitrogen website.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NuPAGE+technical+guide.">NuPAGE technical guide.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+Manual,+Mini+Trans-Blot+Cell.">Instruction Manual, Mini Trans-Blot Cell.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECL+Plus+Western+blotting+detection+reagents+instruction.">ECL Plus Western blotting detection reagents instruction.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+protein+molecular+weights+by+SDS+gel+electrophoresis,+GE+Healthcare+Website.">Estimation of protein molecular weights by SDS gel electrophoresis, GE Healthcare Website.</a> , .</li><li>Yeromin, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+the+CRAC+channel+by+altered+ion+selectivity+in+a+mutant+of+Orai.">Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.</a> Nature. 443, 226-229 (2006).</li><li>Zhang, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-dependent+and+-independent+modes+regulating+CRAC+channel+activity+of+human+Orai1+and+Orai3.">Store-dependent and -independent modes regulating CRAC channel activity of human Orai1 and Orai3.</a> J. Biol. Chem. 283, 17662-17671 (2008).</li><li>Penna, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRAC+channel+consists+of+a+tetramer+formed+by+Stim-induced+dimerization+of+Orai+dimers.">The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.</a> Nature. , .</li></ol>

这里介绍的程序使用双三凝胶与MES作为使用Invitrogen公司NuPAGE NOVEX电泳系统变性PAGE的例子运行缓冲区。此外，这个预制凝胶系统允许下变性或非变性条件以及蛋白质分离，容纳了广泛的分子量范围（1-200 kDa的双三凝胶为36-400 kDa的三醋酸凝胶）。根据你的实验，你可以选择只遵循此处描述的Western印迹技术的一部分，使用直接凝胶染色程序（例如，银或考马斯染色）或其他免疫检测方法（例如，比色法或荧光）。 </...

western blotting using the invitrogen nupage novex bis tris minigels

免疫印迹（或免疫印迹）是一个标准的实验室程序，调查核实的一种蛋白质的表达，确定不同样品中的蛋白质目前的相对量，和免疫共沉淀实验分析结果。在此方法中，检测到目标蛋白质与特定抗体在组织匀浆或提取样品。根据分子量的蛋白质的分离是通过使用变性的SDS - PAGE。转移到膜后，靶蛋白的探测与特定的抗体，通过化学发光法检测。 自第一次描述，Western印迹技术已发生了一些改进，包括预制凝胶和用户友好的设备。在我们的实验室中，我们选择使用自Invitrogen市售NuPAGE电泳系统。这是一个创新的中性pH值，不连续的SDS - PAGE，预制小凝胶体系。该系统提出了一个较长的保质期从8个月至1年不等的预制凝胶ii）一个广泛的分离范围从1到400 kDa的分子量不同的类型，包括传统Laemmli技术的几个优势：一）凝胶使用;及iii）更大的灵活性（丙烯酰胺的比例，凝胶型，和运行缓冲液的离子组成范围）。 在这个视频文章中描述的程序利用了4-12％，二，三梯度凝胶和MES运行说明如何使用Invitrogen公司NuPAGE电泳系统进行免疫印迹的缓冲区，双三不连续缓冲系统。在我们的实验室中，我们已取得了良好的和可重复的结果，采用Western印迹方法的各种生化应用。

Watch this Scientific Journal Video about Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels at JoVE.com

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

这篇技术文章介绍了一个标准的程序，使用市售NuPAGE电泳迷你凝胶体系自Invitrogen Western印迹。

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 ...

Research

JoVE Journal

Biology

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

内淋巴管闭塞手术诱导的内淋巴积水

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

科研

生物学

Western Blotting: Sample Preparation to Detection

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 ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

一个官能二肽的固相合成使用“安全锁扣”方法论

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

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

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

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

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 ...