IN-GEL TRYPTIC DIGESTION FOR SILVER STAINED GELS

Solutions:

• 0.1% TFA/60% acetonitrile.
  
  
• 30 mM potassium ferricyanide (K3 [Fe (CN)6 ], FW 329.3) in water (9.88 mg/ 1ml H2O).
  
  
• 100 mM sodium thiosulfate (Na2S2O3   5H2O, FW 248.2) in water (24.8 mg/ 1ml H2O).
  
  
• 200 mM ammonium bicarbonate (NH4HCO3 FW 79) in water (15.8 mg/ 1ml H2O).
  
  
• 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% acetonitrile / 30 mM NEM acetate pH 8.6.
  
  
• 30 mM NEM acetate (pH8.6).
  
  
• modified Trypsin (Promega).
  

Procedure:

Follow steps 1 and 2 from Coomassie Blue in-gel Tryptic digestion protocol.

1. Wash gel pieces with 250 µl of H2O for 10 minutes at room temperature on tilt table.
   
   
2. Discard wash.
   
   
3. Mix 30 mM potassium ferricyanide and 100 mM sodium thiosulfate 1:1 to make the destaining solution, then add 30-50 µl of the destaining solution to gel pieces; the gel will typically destain in less than 5 minutes and the brownish color disappears.
   
   
4. Remove the destaining solution and wash the gel piece with water until no yellow color is visible (usually 5-6 times).
   

Resume the tryptic digestion protocol for Coomassie Blue stained gels at step 7