Invitrosol digestion protocol
From Proteomics Wiki
Protocol for Trypsin Digest using 80%ACN and Invitrosol
1.Add some buffer (i.e.100mM Tris-HCl, pH 8) to the sample so the sample volume is 100ul. 2.Add 400ul of MeOH and 100ul of Chloroform to the sample. 3.Vortex and spin at full speed for 5 minutes. 4.Discard the supernatant carefully so you don’t disturb the pellet. 5.Add 300ul of MeOH to the pellet and repeat step 3-4. 6.Discard as much supernatant as possible without disturbing the protein pellet. 7.Add 5ul of 5X invitrosol to the pellet and gently resuspend the pellet. 8.Heat the sample at 60oC for 5 minutes. 9.Cool the sample down to room temperature and add 45ul of 90% acetonitrile to the sample (so the final concentration of acentonitrile is ~ 80%). 10.Resuspend gently and sonicate for 2 hours in the water bath. 11.Cool down the sample to room temperature and add trypsin (1:50). 12.Place the sample in the 37oC shaker and digest over night. 13.Next day, add 10ul of 90% Formic acid and speedvac to almost dry. 14.Resuspend the peptides in desired volume of buffer A and load on the column.