Table 18.

Proteolytic Digestion

Processing time	Step	Comments / Tips
15–30 minutes + 4 hours shaking time	Reconstitute trypsin in 200 μL 0.01% acetic acid to stabilize protease and yield a 0.1 μg/μL protease concentration. Vortex.	Wrap a small amount of parafilm around tops of filtration units to prevent them from popping open during the 4-hour digestion period. Remove the parafilm before centrifugation (following digestion).
	Add 70 μL 50 mM ABC buffer to each filtration unit.
	Add 10 μL trypsin solution directly to ABC volume on top of filter for a 1:50 enzyme to protein ratio. Do not pipette on to the sides of the filter.
	Shake filtration units at 600 rpm at 37°C for 4 hours on Isotemp Shaker.