Pulse Chase of Suspension Cells

Materials and Reagents

1. Pulse chase
   1. Cells growing in suspension
      
   2. HBSS (Life Technologies, Invitrogen^TM, catalog number: 14175-095 )
      
   3. RPMI without methionine and cysteine (Sigma-Aldrich, catalog number: R7513 )
      
   4. Dialyzed FBS (Life Technologies, Invitrogen^TM, catalog number: 26400044 )
      
   5. N-Ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: R3876 )
      
   6. Cyclohexamide (CHX) (Sigma-Aldrich, catalog number: C7698 )
      
   7. Express ³⁵S protein labelling mix (Perkin Elmer, catalog number: NEG072014MC )
      
   8. Methionine (Sigma-Aldrich, catalog number: M5308 )
      
   9. Cysteine (Sigma-Aldrich, catalog number: C7352 )
      
   10. Labeling medium (see Recipes)
       
   11. Chase medium (see Recipes)
       
   12. 2x stop buffer (see Recipes)
       
   
   
2. Cell lysis and immunoprecipitation
   1. Antibody against protein of interest
      
   2. Protein A/G beads (Thermo Fisher Scientific, catalog number: 20422 )
      
   3. Complete Protease Inhibitor Tablets (Roche Diagnostics, catalog number: 11836145001 )
      
   4. Lysis buffer (see Recipes)
   
   
3. SDS-PAGE
   1. 4-12% Bis-Tris protein gel (Life Technologies, Invitrogen^TM)
      
   2. MOPS running buffer (Life Technologies, Invitrogen^TM, catalog number: NP0001 )
      
   3. Amplify solution (GE Healthcare, catalog number: NAMP100 )
      
   4. Gel drying solution (Life Technologies, Invitrogen^TM, catalog number: LC4025 )
      
   5. Gel fixing solution (see Recipes)

Equipment

1. Incubator
   
2. Eppendorf tube
   
3. 26-gauge needle
   
4. 1 ml syringe
   
5. Heat block
   
6. Gel dryer
   
7. Phosphor imaging screen
   

Procedure