Protein Chemistry Laboratory

On-PVDF membrane digestion procedure for obtaining internal peptide sequence information:

On PVDF Digestion:

All tubes and pipette tips rinsed with 0.1% TFA/50% ACCN prior to use!

1. Excise bands and cut into 1 mm x 2mm pieces.
2. Pre-wet with MEOH and place into microcentrifuge tube.
3. Destain for 1 min with 0.1% TFA/50% ACCN
4. Rinse 5X with H2O. Sonicate 5 min per wash.
5. Add 50µl digestion buffer to PVDF
-digestion buffer = (100 mM TRIS, 1% Reduced Triton X-100, 10% ACCN, pH 8.0)
6. Add 5µl 45 mM DTT and incubate 55^o C for 30 min.
7. Add 5µl 100 mM Iodoacetic acid and incubate 30 min @ room temp in dark.
8. Remove digestion buffer and wash 1X with 50µl digesiton buffer.
9. Add 50µl digestion buffer to PVDF.
10. Add 2µ Trypsin (Modified Trypsin-Promega) at 0.1 mg/ml.
11. Incubate 37^o C for 4 hrs.
12. Add another 2µl Trypsin (0.1mg/ml).
13. Digest overnight at 37^o C.
14. Sonicate sample for 5 min, centrifuge and remove supernatant (save).
18. Extract again with 25µl of 0.1% TFA/50% ACCN by sonicating for 5 min.
19. Centrifuge and save supernatant.
20. Extract again with 25µl of 0.1% TFA by sonicating for 5 min.
21. Centrifuge and save supernatant.
22. Pool all saved supernatants and store at -20^o C. The sample is now ready for separation on the 173A Microblotter.