Protein ID Solutions Sample Prep Guide
73
In-Solution Tryptic Digestion
7
Quenching of excess IAM
1 Add 1.0 µL DTT stock solution to destroy excess IAM.
2 Allow to stand for 1 hour in the dark (foil-covered rack) at room
temperature.
Dilution and pH adjustment
1 Add 300 µL water to dilute denaturant.
2 Add 100 µL ammonium bicarbonate stock solution to raise pH.
3 Optionally check pH by placing 0.5 to 1 µL on a strip of pH indicator paper.
Typical value is 7.5 to 8.0. It is more important to check pH when the pH of
the starting sample is unknown.
4 Add more base (ammonium bicarbonate) if pH is not in the 7 to 9 range.
Digestion
1 Make fresh stock solution of trypsin in trypsin storage solution, as
described on
page 75
. Allow 15 min for complete re-suspension.
2 If you plan to digest less than 20 µg total protein, prepare trypsin
intermediate solution as described on
page 76
.
C A U T I O N
If IAM is not destroyed, it will slowly alkylate lysines.
C A U T I O N
Dilution is important because trypsin will be denatured if TFE level is greater than 5%. If
urea or guanidine HCl are used as denaturants, trypsin will be denatured if their levels
are greater than about 1M.
pH adjustment is important because trypsin loses activity if the pH is not in the 7 to 9
range.
N O T E
When handling a large batch of samples, combine water and ammonium bicarbonate stock
(steps 1 and 2) to make a 25 mM solution and pipette 400 µL of the resulting solution.