IN-GEL TRYPTIC DIGESTION
FOR GELS STAINED WITH COOMASSIE BLUE

Solutions:

• 0.1% TFA/60% CH3CN
  
  
• 50% H2O/ 50% CH3CN
  
  
• 1 M NH4HCO3 (stock solution)
  
  
• 50% CH3CN / 50 mM NH4HCO3
  
  
• 100 mM NEM acetate pH 8.6 (N-Ethyl Morpholine, Aldrich stock = 7.86 M prepare fresh; adjust pH to 8.6 with 1M acetic acid)
  
  
• 50% CH3CN / 30 mM NEM acetate pH 8.6
  
  
• 30 mM NEM acetate pH 8.6
  
  
• Trypsin (modified, Promega)

Procedure:

1. Prewash 1.5 ml Eppendorf tubes with 3 X500 µl of 0.1% TFA/60% CH3CN.
   
   
2. Cut the sample gel pieces, a blank gel piece, and a control protein gel piece into roughly 1 mm3 cubes and put them into prewashed tubes separately.
   
   
3. Add 2X250 µl of 50% H2O/ 50% CH3CN and wash for 30 minutes.
   
   
4. Remove wash.
   
   
5. Add 250 µl of 50% CH3CN / 50 mM NH4HCO3 to all samples.
   
   
6. Wash for 30 minutes at room temp on tilt table.
   
   
7. Remove wash.
   
   
8. Add 3X250 µl of 50% CH3CN / 30 mM NEM acetate pH 8.6 to all samples.
   
   
9. Wash for 30 minutes at room temp on tilt table.
   
   
10. Remove wash.
    
    
11. SPEEDVAC gel pieces to COMPLETE dryness.
    
    
12. a) Add 0.1 µg modified trypsin (Promega) per 15 mm3 of sample gel in 15 µl of 30 mM NEM acetate pH 8.6.
    
    b) Let stand for 5-10 minutes to allow enzyme/buffer to absorb into gel.
    
    
13. Add an additional 10-15 µl of 30mM NEM acetate pH 8.6 that does not contain enzyme to just cover the gel pieces.
    
    
14. Incubate overnight at 37°C.
    
    
15. Extract the peptides 2x by adding 50 µl of 0.1% TFA/60% CH3CN and shaking at room temp for 30 min.
    
    
16. Transfer the extracts to a siliconized tube prewashed 3X with 500 µl of 0.1% TFA/60% CH3CN.
    
    
17. Speedvac to dryness for subsequent MS analysis.