i have, in the past, used the following basic recipe to get protein
bands out of PAGE gels for HPLC (and later MS) analysis:
1. excise the band after sample elucidation (ie autorad, staining)
2. wash the slices 2x5 min in 50% methanol
3. wash in 1x5 min with ddH2O
4. add 0.5ml ddH2O and place on a rocker overnight at 4'C
5. spin samples at 14k, 10 minutes in microfuge
6. remove supernatant and spin this down again for 10 min at 14k
7. dry this supernatant down
8. resuspend in 100ul 50% formic acid, redry
hope this helps.
another procedure i used was (again in an eppendorf, prep for HPC
analysis):
1. take gel slices and add 1ml 50% MeOH, rocker at RT for 1h
2. wash with HPLC water for 30 min
3. wash twice with 1ml 50 mM Ammonium Bicarb (in HPLC water) 5 min
4. add 0.5 ml 50 mM Ammonium Bicarb (in HPLC water), 37'C
5. add 20 ul 1mg/ml trypsin, incubate for 4h at 37'C. repeat four more
times
6. remove gel band, dry down sample (speed vac), keep at -20'C until use
if you didn't excise enough protein, repeat (the gel bands can be kept
at -20'C for a while).
hope this helps,
jose nazario jose at cwru.edu
