IN-GEL TRYPTIC DIGESTION IN
96-WELL MICROTITER PLATE
SILVER STAINED GELS

96-Well Microplate: Nunc

V-96 Polypropylene Plate Purchased from Fisher Scientific. Cat No. 12-565-263

Special Pipetter: Multichannel Pipetter

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% CH3CN / 30 mM NEM acetate pH 8.6.
  
  
• 30 mM NEM acetate (pH8.6).
  
  
• modified Trypsin (Promega)

Procedure:

Follow steps 1 and 2 for in-gel tryptic digestion in a 96-well microtiter plate for Coomassie blue stained gels.

1. Wash gel pieces with 150 µl of H2O for 10 minutes at room temperature on tilt table.
   
   
2. Remove wash.
   
   
3. Mix 30mM potassium ferricyanide and 100mM sodium thiosulfate 1:1 to make the destaining solution, then add 30-50 µl of the destaining solution to gel piece; the gel will destain in less than 5 min and the brownish color disappears.
   
   
4. Remove the destaining solution and wash the gel piece with water until no yellow color remains (usually 5-6 times).
   
   
5. Wash the gel pieces with 150 µl of 200 mM ammonium bicarbonate for 20 minutes at room temperature on tilt table.
   
   

Resume the microtiter plate tryptic digestion protocol for Coomassie Blue stained gels at step 8.