Trypsin Digestion
Make 6M Urea solution by adding 108.11 mg of Urea to 300 µL of 50mM ammonium bicarbonate ( NH4HCO3 ) containing protein .
Add 20 µL of 1.5 M Tris pH 8.8 .
Add 7.5 µL of 200mM TCEP and incubate for 1 hour at 37°C .
Add 60 µL of 200mM iodoacetamide and vortex .
Incubate for one hour at room temperature in a dark area .
Add 60 µL of 200mM DTT and vortex .
Incubate for one hour at room temperature .
Aliquot 150 µL each into three 1.5 mL centrifuge tubes .
Add 800 µL of 25mM ammonium bicarbonate to each tube to dilute the urea .
Add 200 µL of MeOH to each tube .
Add Trypsin to protein at a ratio of 50 protein to 1 trypsin and incubate overnight at room temperature .
Speedvac until dry .
Add 200 µL of molecular biology grade water to tubes three times and speedvac to reduce the ammonium bicarbonate .
Store at - 80°C .
