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.
