Materials and Reagents

1. HeLa S3 and HeLa cells were cultured in Dulbecco’s modified Eagles Medium containing 10% fetal bovine serum (Invitrogen) and antibiotics
   
2. Protein A beads (Bio-Rad Laboratories); Protein G beads (Santa Cruz Biotechnology)
   Note: For rabbit polyclonal antibodies and mouse monoclonal antibodies IgG2a, IgG2b, IgG3, we usually couple antibodies to protein A beads; For mouse monoclonal antibodies IgG1, rat monoclonal antibodies, and mouse, rat, goat, and donkey polyclonal antibodies, we usually couple antibodies to protein G beads.
   
3. Bifunctional cross-linker dimethyl pimelimidate (DMP) (MW: 259.177) (Sigma-Aldrich, catalog number: D8388 )
   
4. Phosphate buffered saline (PBS)
   
5. Tween 20
   
6. Triton X-100
   
7. Sodium borate (pH 9.0)
   
8. Hepes
   
9. KCl
   
10. NaN₃
    
11. NP-40
    
12. Glycerol
    
13. EGTA
    
14. MgCl₂
    
15. DTT
    
16. Microcystin
    
17. Leupeptin
    
18. Pepstatin
    
19. Chymostatin
    
20. β-mercaptoethanol
    
21. Bromophenol blue
    
22. PBST buffer (see Recipes)
    
23. Lysis buffer (see Recipes)
    
24. 1x SDS protein gel sample loading buffer (see Recipes)
    

Equipment

1. Hematology/chemistry mixer (Fisher Scientific)
   
2. Centrifuge (Eppendorf 5415D centrifuge)
   
3. Incubator
   

Procedure