Protein Translation Study – Label Protein with S35 Methionine in Cells

Materials and Reagents

1. Methionine-free medium DMEM (Sigma-Aldrich, catalog number: D0422 )
   
2. Fetal calf serum (Hyclone, catalog number: SV30160.03 )
   
3. Fetal bovine serum (FBS)
   
4. Penicillin/streptomycin/glutamine
   
5. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 10010-056 )
   
6. Protein assay kit (DC Protein Assay Kit I-500) (Bio-Rad, catalog number: 0111EDU )
   
7. Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )
   
8. EasyTaq -[³⁵S]-Methionine, 5 mCi (185 MBq), stabilized aqueous solution (Perkinelmer, catalog number: NEG709A005MC )
   
9. Hybond ECL Nitrocellulose Membrane (Amersham, catalog number: RPN68D )
   
10. Kodak Biomax XAR film (Sigma-Aldrich, catalog number: F5763 )
    
11. Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, catalog number: WBKLS0500 )
    
12. Anti-MDM2 (Santa Cruz)
    
13. HEPES
    
14. NaCl
    
15. Glycerol
    
16. Triton X-100
    
17. MgCl₂
    
18. EGTA
    
19. Na₄P₂O₇
    
20. NaF
    
21. Aprotinin
    
22. Leupeptin
    
23. PMSF
    
24. Na₃VO₄
    
25. 2-mercaptoethanol
    
26. Acrylamide
    
27. Bromophenol blue
    
28. Ammonium persulfate (APS)
    
29. Lysis buffer (see Recipes)
    
30. HNTG buffer (see Recipes) 
31. Protein A or G agarose beads (see Recipes)
    
32. 2x laemmli buffer (see Recipes)
33. SDS-polyacrylamide gel (see Recipes)
    
34. 10x electrophoresis buffer (see Recipes)
    
35. 1x transfer buffer (see Recipes)
    

Equipment

1. Centrifuges
   
2. Vortexer
   
3. Tissue culture hood and incubator
   
4. Radioactive material and room
   
5. Western-Blot apparatus
   
6. Developer
   
7. Hamilton syringe
   
8. Spectrophotometer
   
9. Rocker
   
10. T25 flask
    

Procedure