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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 Jian-Guo Zhang
JB Jake Baum
Mireille H. Lahoud Mireille H. Lahoud
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: 21698

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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) search F-actin (F-肌动蛋白) search Dendritic Cells (树突状细胞) search Clec (中国学习者英语语料库) search

Materials and Reagents

  1. Rabbit muscle G-actin (Cytoskeleton, catalog number: AKL95-C )
  2. Recombinant Clec9A ectodomain was expressed and purified by affinity chromatography and size-exclusion chromatography as described previously 5
  3. Tris
  4. CaCl2
  5. ATP
  6. β-mercaptoethanol
  7. KCl
  8. MgCl2
  9. NuPAGE Novex 4-12% Bis-Tris gel (Life Technologies, Invitrogen™, catalog number: NP0327BOX )
  10. Laemmli reducing SDS-PAGE sample buffer (Laemmli 2x concentrate) (Sigma-Aldrich, catalog number: S3401 )
  11. Precision Plus Protein molecular weight marker (Bio-Rad Laboratories, catalog number: 161-0373 )
  12. Coomassie Brilliant Blue
  13. 1x G-buffer (see Recipes)
  14. 10x F-buffer (see Recipes)

Equipment

  1. Superdex S200 10/300 GL column (GE Healthcare)
  2. AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare)
  3. TLA 100 rotor and Beckman Coulter Optima TL Ultracentrifuge (Beckman Coulter)
  4. Polycarbonate centrifuge tubes (7 x 20 mm, 230 μl) (Beckman Coulter, catalog number: 343775 )
  5. 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

© 2012 The Authors; exclusive licensee Bio-protocol LLC.

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.

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分类

生物化学 > 蛋白质 > 相互作用 > 蛋白质-蛋白质相互作用

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