Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Materials and Reagents

1. PBS
2. HEPES
3. KOAc*
4. Mg(OAc)₂
5. CaCl₂
   
6. Sorbitol
   
7. Wash buffer
8. EDTA free protease inhibitors (Roche Diagnostics)
   
9. Digitonin (EMD Chemicals)
10. Protease Inhibitors (DMSO, leupeptin, pepstatin) (Sigma-Aldrich)
11. ANTI-Flag M2 affinity gel (Sigma-Aldrich)
    
12. 3x FLAG peptide (Sigma-Aldrich)
    
13. NaF^# (Ser/Thr phosphatase inhibitor)
    
14. Na₃VO₄ (Tyr phosphatase inhibitor)
    
15. Lysis buffer (see Recipes)
    
16. Immunoprecipitation buffer (see Recipes)
17. Elution buffer (see Recipes)

Equipment

1. BECKMAN centrifuge and TLA-55 rotor
2. Acid washed 425-600 glass beads (Sigma-Aldrich)
3. Beckman centrifuge tubes (Beckman Coulter)
   

Procedure