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Crosslinking and Immunoprecipitation in Zebrafish

LJ Lili Jing

Published: Jan 20, 2012 DOI: 10.21769/BioProtoc.174 Views: 14535

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Abstract

Immunoprecipitation (IP) is a routine method to detect protein binding and interactions. But the weak binding between two proteins is often hard to detect during regular IP procedure. This protocol offers a crossliking and IP combination method to detect weak binding of proteins in zebrafish embryos.

Materials and Reagents

  1. NaCl
  2. KCl
  3. CaCl2
  4. Tris
  5. EDTA
  6. Glycerol
  7. Triton X-100
  8. TBST
  9. Glycine
  10. NP-40
  11. Na deoxycholate
  12. SDS
  13. DTSSP (Thermo Fisher Scientific, catalog number: 21578 )
  14. HEPES (Life Technologies, Invitrogen™, catalog number: 15630 )
  15. Protease inhibitors (Sigma-Aldrich, catalog number: P2714 )
  16. Anti-HA affinity matrix (Roche Diagnostics, catalog number: 11815016001 )
  17. Anti-FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220 )
  18. Modified Ringer’s solution (see Recipes)
  19. Lysis buffer (see Recipes)
  20. RIPA (see Recipes)
  21. Wash buffer (see Recipes)

Equipment

  1. Kontes tubes

Procedure

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

Category

Biochemistry > Protein > Immunodetection

Biochemistry > Protein > Interaction

Biochemistry > Protein > Interaction

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