Materials and Reagents

1. HeLa cells (ATCC, catalog number: CCL-2 )
   
2. SBP-Calmodulin Tag Expression Vector pNTAP (Inter Play N-Terminal Mammalian TAP Vector, Agilent Technologies-Stratagene, catalog number: 240101 )
   
3. Lipofectamine (Life Technologies, Invitrogen^TM, catalog number: 11668-027 )
   
4. Fugene 6 (F. Hoffmann-La Roche, catalog number: 05061377001 )
   
5. FLAG tag Expression Vector pCMV-Tag2 (Agilent Technologies-Stratagene, catalog number: 211172 )
   
6. NP40
   
7. Steptavidin agarose beads (Sigma-Aldrich, catalog number: S-1638 )
   
8. Anti- Calmodulin antibody (Upstate, catalog number: 07-482 )
   
9. Anti- Flag antibody (Sigma-Aldrich, catalog number: F1804-200UG )
   
10. Protease inhibitor cocktail, EDTA free (F Hoffmann-La Roche, catalog number: 04693159001 )
    
11. DNase I (Sigma-Aldrich, catalog number: DN25 )
    
12. Benzonase nuclease(Sigma-Aldrich, catalog number: E1014 )
    
13. Laemmli loading buffer (see Recipes)
14. Sucrose buffer (see Recipes)
15. Nuclei Lysis buffer for hard to extract nuclear factors (see Recipes)
16. Nuclei Lysis buffer for soluble nuclear factors (see Recipes)
    

Equipment

1. Wheel in a cold room
   
2. Refrigerating centrifuge 1.5 ml tubes
   

Procedure