A general set-up for a digestion that we would analyze by mass spectrometry would be as follows: Re-suspend 20 ug of Trypsin in 200 ul of water with mixing for five minutes. This will give you a working concentration of Trypsin of 100 ng/ul. Add 5 ul of a protein sample containing about 2 ug of total protein to a empty sample tube. Add 20 ul of 1X Trypsin buffer to the tube containing the sample and mix well. We generally do Trypsin reactions with a sample protein: Trypsin ratio by weight of 20:1. The reaction is incubated overnight (18 hrs) at 37 C and then analyzed by MS or MS/MS or stored frozen at -20 C until use.