Tryptic digest of SDS-PAGE Purified proteins.

1. Block membrane – 0.5% PVP-40/100mM Acetic Acid – 30-90’

2. Wash – 5X HPLC water

3. Resuspend lyophilized trypsin in appropriate amount (digest proteins on each NC membrane with enough suspended trypsin to cover membrane, using 20 mg/5 samples) of 200mM ammonium bicarbonate (pH 8.2)/Acetonitrile (95:5)

- O/N at 37⁰C

- Next day, add another aliquot of trypsin in 1/10 the volume to each sample.

8 hrs-O/N at 37⁰C

4. Add 400 ml HPLC water and Speed Vac supe.

5. Resuspend in 50 ml performic acid. ( 9 parts formic acid/ 1 part 30% hydrogen peroxide – incubate @ RT for 30’)

- on ice – 60’

6. Add 400 ml HPLC water and Speed Vac – 2X

7. Inject on HPLC