Name: actin co-sedimentation assay; for the analysis of protein binding to f-actin
Uploaded: 2008-03-28T01:00:00
Description: Watch this Scientific Journal Video about Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin at JoVE.com

感谢您在惠特曼在加州大学旧金山分校的实验室Praveen库马尔博士在HaCaT细胞中的肌动蛋白的电影。

1。肌动蛋白的制备在这个实验中使用的肌动蛋白的来源是从骨架公司股票在10毫克/毫升-70℃冷冻保存等分获得的非肌肉的人血小板（β-肌动蛋白）。肌动蛋白检测，稀释至0.4毫克/毫升含有5毫米的Tris pH值8， 氯化钙 0.2毫米，0.2毫米ATP和0.5毫米的DTT，4℃离心10分钟，在20000克的缓冲上清，这是单体肌动蛋白聚合。另外50 mM的氯化钾，1毫米的ATP和2毫米MgCl 2的诱导肌...

<ol>
	<li>Senetar, M. A., Foster, S. J., McCann, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasteric+inhibition+mediates+the+interaction+of+the+I/LWEQ+module+proteins+Talin1,+Talin2,+Hip1,+and+Hip12+with+actin.">Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin.</a> Biochemistry. 14, 15418-15428 (2004).</li><li>Goldmann, W. H., Hess, D., Isenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+intact+talin+and+talin+tail+fragment+on+actin+filament+dynamics+and+structure+depends+on+pH+and+ionic+strength.">The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.</a> Eur J Biochem. 260, 439-445 (1999).</li><li>Lee, H., Bellin, R. M., Walker, D. L., Patel, B., Powers, P., Liu, H., Garcia-Alvarez, B., de Pereda, J., Liddington, R. C., Volkmann, N., Hanein, D., Critchley, D. R., Robson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+Actin-binding+Site+within+the+Talin+FERM+Domain.">Characterization of an Actin-binding Site within the Talin FERM Domain.</a> J Mol Biol. 22, 771-784 (2004).</li></ol>

肌动蛋白共沉淀是一个简单分析F -肌动蛋白结合specfic蛋白在体外试验。在这个视频中，我们展示了一个简单的肌动蛋白可以进行合作沉淀的一个例子。我们将展示如何准备F -肌动蛋白，准备要测试的蛋白和肌动蛋白的共同沉淀的过程。开展肌动蛋白共同沉淀分析时应该考虑的若干要点。例如，孵化缓冲组件可以进行测试，pH值，温度和盐的浓度可能会影响到F -肌动蛋白结合蛋白的不同而有所不同。我们已经证明?...

actin co-sedimentation assay; for the analysis of protein binding to f-actin

细胞内的肌动蛋白细胞骨架的微丝网络，使细胞和细胞进程的运动，而产生的紧张局势，并有助于保持细胞形态。虽然肌动蛋白细胞骨架是一个刚性的结构，它是一个动态的结构，不断重塑。一些蛋白质可以结合到肌动蛋白细胞骨架。 F - actin的一种特定的蛋白质结合通常需要支持细胞生物学的观察，或进一步了解由于肌动蛋白细胞骨架重塑的动态过程。肌动蛋白共沉淀实验是在体外实验中通常用来分析特定的蛋白质或F -肌动蛋白结构域的结合。检测的基本原则，包括与F -肌动蛋白（孵化全长或域），超速离心沉淀的F -肌动蛋白和合作与F -肌动蛋白沉积的蛋白质的分析步骤。肌动蛋白的共沉淀实验可以设计相应的措施，肌动蛋白的亲和力和竞争分析。

Watch this Scientific Journal Video about Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin at JoVE.com

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

蛋白质结合丝状肌动蛋白通过不同的肌动蛋白结合模块（F - actin的）。在这个视频中，我们共同沉淀，这是一个经常使用的分析，结合F -肌动蛋白的蛋白质或特定域在体外实验证明肌动蛋白的过程。

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Research

JoVE Journal

Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Detection of Protein Ubiquitination

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Linearization of the Bradford Protein Assay

Bradford蛋白含量的线性

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析