Analyze p53 degradation by 35S p53 Pulse Chase Analysis

Materials and Reagents

1. Dulbecco’s Modified Eagle Medium (DMEM) methionine and cysteine free media (1x) (Life Technologies, Invitrogen™, catalog number: 21013-024 )
   
2. Dialysed fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 26400-036 )
   
3. Express ³⁵S protein labelling mix (PerkinElmer, catalog number: NEG 0720008MC )
   
4. Cell lifter (Corning Incorporated, catalog number: 3008 )
   
5. Protein A sepharose (Zymed, catalog number: 10-1042 )
   
6. p53 DO-1 (Santa Cruz, catalog number: SC-126 )
   
7. DC Protein assay kit (Bio-Rad Laboratories, catalog number: 500-0116 )
   
8. Bio-Rad safe stain solution (Bio-Rad Laboratories, catalog number: 161-0786 )
   
9. L-glutatmine or Glutamax (Life Technologies, Invitrogen™, catalog number: 35050-061 )
   
10. cOmplete Ultra tablets (Roche Applied Science, catalog number: 05 892 988 001 )
    
11. PhosSTOP tablets (Roche Applied Science, catalog number: 049068450001 )
    
12. NP-40 (IGEPAL, catalog number: CA-630 )
    
13. Beta mercapotethanol
    
14. Bromophenol blue
15. Complete media (see Recipes)
    
16. Starvation media (see Recipes) 
17. Dulbecco’s PBS (see Recipes)
    
18. 1x lysis buffer (see Recipes)
    
19. 2x SLB buffer (see Recipes)
20. Washed protein A sepharose (see Recipes)
    

Equipment

1. Bioruptor sonicator (Diagenode)
   
2. Macsmix tube rotator (Miltenyi Biotech GmbH) or standard Rotating wheel
   
3. Heat block
   
4. SDS–polyacrylamide gel electrophoresis (PAGE) running equipment
   
5. Orbital shaker
   
6. 3 mm whatman paper and plastic wrap
   
7. Gel Dryer and pump
   
8. Storage phosphor screen (Molecular Dynamics) and Typhoon Trio (GE Healthcare)
   

Software

1. Image Quant software (Molecular Dynamics)

Procedure