In Gel Digestion:
1. Cut the gel section containing the protein of interest
into 1 x 2 mm pieces and place in an eppendorf tube. Repeat
for the blank section of gel.
2. Add 250Ál 200 mM NH4HCO3 / 50% CH3CN
to the gel pieces. (REMOVES CB)
3. Wash for 30 minutes at room temperature on a rocker table.
4. Remove wash and SAVE.
5. Add 100Ál NH4HCO3 / 50% CH3CN
to the gel pieces.
6. Calculate the volume of 45 mM DTT needed to give a final
[DTT] of 1 mM in the sample.
7. Add the above volumes of DTT and then incubate at 37oC
for 20 minutes.
8. After incubation, add an equal volume of 100 mM iodoacetic
acid or 200 mM MNS (methyl 4-nitrobenzene sulfonate) as DTT.
9. For carboxymethyl cysteine, incubate in the dark for 20
minutes. For s-methyl cysteine (MNS treatment), incubate at
37oC for 40 minutes.
10. After incubation, remove supernatant and SAVE.
11. Repeat steps 2-4.
12. Speedvac gel pieces to complete dryness.
13. Immediately before use, make up a 0.033 mg/ml enzyme
stock solution in 200 mM NH4HCO3 by
mixing 5.0 Ál of 0.1 mg/ml enzyme stock with 10.0 Ál 200 mM
14. Add a volume of 0.033 mg/ml trypsin or lysyl
endopeptidase that equals the initial gel volume (ie. 15Ál
per 15 mm3 of gel.
15. Incubate 37oC for 15 minutes. (LOOK
TO MAKE SURE GEL IS TOTALLY IMMERSED).
16. If the gel pieces are not totally immersed, add
additional enzyme solution until they are just covered.
17. Incubate at 37oC for 24 hours.
18. Extract peptides by adding at least 100Ál 0.1% TFA, 60%
CH3CN and shaking on a rocker table at room
temperature for 60 minutes.
19. Remove supernatant containing peptides and repeat steps
18 and 19.
20. Speedvac dry the combined washes.
21. Redissolve the dried peptides in 20Ál 0.05% TFA / 25% CH3CN.
22. Sample is ready for reverse phase HPLC separation of
peptides on the 173A Microblotter.