Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Materials and Reagents

1. EDTA free protease inhibitors (Roche Diagnostics)
2. Digitonin (EMD Chemicals)
   
3. Protease Inhibitors (DMSO, leupeptin, pepstatin) (Sigma-Aldrich)
4. ANTI-Flag M2 affinity gel (Sigma-Aldrich)
5. 3x FLAG peptide (Sigma-Aldrich)
   
6. NaF# (Ser/Thr phosphatase inhibitor)
7. Na3VO4 (Tyr phosphatase inhibitor)
8. HEPES
9. KOAc
10. Mg(OAc)₂
11. CaCl₂
    
12. Sorbitol
13. Glycerol
14. KOH
15. PMSF
16. DMSO
17. Lysis buffer (see Recipes)
18. Immunoprecipitation buffer (see Recipes)
19. 50 ml lysis buffer (see Recipes) 
20. 14 ml IP buffer with 2% digitonin (see Recipes)
21. 20 ml IP buffer with 0.1% digitonin (see Recipes)
22. 40 ml IP buffer with 0.1% digitonin (see Recipes)
23. 3x FLAG elution buffer (see Recipes)
    

Equipment

1. Avestin Emulsiflex C3 homogenizer (Avestin^®)
2. Beckman centrifuge and Type 70 Ti rotor (Beckman Coulter) 
3. Beckman polycarbonate centrifuge tubes (Beckman Coulter, catalog number: 355631 )
4. 1 L centrifuge bottles
5. 50 ml conical tubes
   

Procedure