Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

TAP Purification of Yeast Proteins

Bio-protocol Editor Bio-protocol Editor

Published: Jan 5, 2011 DOI: 10.21769/BioProtoc.17 Views: 17504

pdf PDF PDF
ask Ask a question
How to cite
Favorite
Cited by

Abstract

Tandem affinity purification (TAP) is used to look at protein-protein interaction. Its use relies on generating a fusion protein with a TAP tag on the C- or N- terminal end. In this protocol, a two-step purification of N-terminus TAP-tagged proteins from yeast is described.

Materials and Reagents

  1. Complete Protease Inhibitor Cocktail Tablet (Roche Diagnostics)
  2. 100% NP-40 (Sigma-Aldrich)
  3. AcTEV Protease (Life Technologies, Invitrogen™)
  4. IgG Sepharose 6 Fast Flow (GE Healthcare Life Science)
  5. Calmodulin affinity resin (Guidechem/Stratagene)
  6. SDS lysis buffer
  7. Beta-ME
  8. Leupeptin
  9. Mg2 acetate
  10. Acetone
  11. BME
  12. EDTA
  13. CaCl2
  14. NaF
  15. Imidazole
  16. NP40 buffer (see Recipes)
  17. IPP150 buffer (see Recipes)
  18. IPP150 calmodulin binding buffer (see Recipes)
  19. TEV cleavage buffer (see Recipes)
  20. IPP150 calmodulin elution buffer (see Recipes)

Equipment

  1. Avestin Homogenizer (Avestin®)
  2. BECKMAN centrifuge and rotor (BECKMAN Coulter)
  3. Polycarbonate tubes
  4. Chromatography column
  5. Microfuge tube
  6. Shaker

Procedure

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

Category

Biochemistry > Protein > Isolation and purification

Microbiology > Microbial biochemistry > Protein

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A