共沉降法检测纤维状肌动蛋白的直接结合

Wilson  Wong; Jian-Guo Zhang; Jake Baum; Mireille H. Lahoud; Ken Shortman

doi:10.21769/BioProtoc.270

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Co-sedimentation Assay for the Detection of Direct Binding to F-actin

共沉降法检测纤维状肌动蛋白的直接结合

WW Wilson Wong

Jian-Guo Zhang

JB Jake Baum

Mireille H. Lahoud email

KS Ken Shortman

发布: 2012年10月05日第2卷第19期 DOI: 10.21769/BioProtoc.270 浏览次数: 20667

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

Apr 2012

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

蛋白质pull-down免疫共沉淀实验分析病毒DNA结合蛋白质之间的直接相互作用

Ana Lechuga [...] Modesto Redrejo-Rodríguez

2018年01月05日 11080 阅读

采用Förster共振能量转移法检测大肠埃希杆菌细胞质和周质中蛋白质的相互作用

Nils Y. Meiresonne [...] Tanneke den Blaauwen

2018年01月20日 13505 阅读

使用 Spy Chemistry 进行抗体纯化

Xiaofeng Yang [...] Zisha Lao

2025年04月20日 1036 阅读

Abstract

This protocol describes measurement of direct protein-protein interactions by actin co-sedimentation assay. The actin co-sedimentation assay is a well-established technique and has been commonly used to demonstrate binding of proteins that interact directly with actin filaments (Ahrens et al., 2012; Mehta and Sibley, 2010; Schuler et al., 2005; Singh et al., 2011). We and others have previously shown that the damaged cell-recognition molecule C-type lectin 9A (Clec9A) recognises a conserved component within nucleated and non-nucleated cells that is exposed only when the cell membrane is damaged (Srivastava and Barber, 2008; Zhang et al., 2012). Here we use Clec9A as an example and present a detailed procedure for demonstrating the direct binding of purified recombinant Clec9A ectodomain to actin filaments.

Keywords: Actin (肌动蛋白)

F-actin (F-肌动蛋白)

Dendritic Cells (树突状细胞)

Clec (中国学习者英语语料库)

Materials and Reagents

Rabbit muscle G-actin (Cytoskeleton, catalog number: AKL95-C )
Recombinant Clec9A ectodomain was expressed and purified by affinity chromatography and size-exclusion chromatography as described previously 5
Tris
CaCl₂
ATP
β-mercaptoethanol
KCl
MgCl₂
NuPAGE Novex 4-12% Bis-Tris gel (Life Technologies, Invitrogen™, catalog number: NP0327BOX )
Laemmli reducing SDS-PAGE sample buffer (Laemmli 2x concentrate) (Sigma-Aldrich, catalog number: S3401 )
Precision Plus Protein molecular weight marker (Bio-Rad Laboratories, catalog number: 161-0373 )
Coomassie Brilliant Blue
1x G-buffer (see Recipes)
10x F-buffer (see Recipes)

Equipment

Superdex S200 10/300 GL column (GE Healthcare)
AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare)
TLA 100 rotor and Beckman Coulter Optima TL Ultracentrifuge (Beckman Coulter)
Polycarbonate centrifuge tubes (7 x 20 mm, 230 μl) (Beckman Coulter, catalog number: 343775 )
GS-800 calibrated densitometer and Quantity one image analysis software (Transmission densitometer) (Bio-Rad Laboratories, catalog number: 1707980 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Wong, W., Zhang, J., Baum, J., Lahoud, M. H. and Shortman, K. (2012). Co-sedimentation Assay for the Detection of Direct Binding to F-actin. Bio-protocol 2(19): e270. DOI: 10.21769/BioProtoc.270.