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

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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

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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

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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.