Gel slice digestion of silver-stained gels
To destain:
Rinse gel with water before cutting and putting into tubes.
Solutions:
30mM K3Fe(CN)6, 0.010g/mL;
100mM AMBIC
100mM Na2S2O3, 0.016g/mL;
-Wash gel pieces thoroughly with water
-Mix reducers 1:1 and add to gel pieces to cover them
-Vortex until brown color disappears (~10 min)
-Remove solution
-Wash with 100mM AMBIC to remove yellow tint, replace as needed. Yellow must be
gone.
To prepare slices:
Pull off solution and add 50/50 (v/v) AcN/100 mM AMBIC and incubate 15 min.
Pull off solution and add 100% AcN and incubate on shaker 15 min until gel slices are
white and sticky.
Pull off solution and cover with 100 mM AMBIC and incubate 5 min. Add equal volume
of AcN and incubate 15 min.
Pull off solution and dry gel slices in speed-vac (must be very dry!).
Reduction/alkylation:
Cover dried gel slices with 10 mM dithiothreitol/100 mM AMBIC and incubate at 56 C
for 45 min.
Remove solution and cover gel slices with 25 mM iodoacetamide/100 mM AMBIC.
Incubate at room temp for 60 min in the dark.
Remove solution and add 100 mM AMBIC. Wash for 5 min.
Remove solution and add 50/50 (v/v) 100 AMBIC/AcN. Incubate at RT for 15 min.
Remove solution and dry gel slices in speed-vac.
Digestion:
Rehydrate in trypsin solution (12.5 ng/uL in 50mM AMBIC- keep reagents at 4 C) on ice
for 1 hour.
Pull off excess and cover gels with 50 mM AMBIC and incubate at 37 C overnight.
Pull off supernatant and MALDI 0.5uL if needed.
For LC-MS/MS, acidify supernatant to 1% Acetic acid. Add enough 0.1% TFA to cover
gel and incubate 15 min on shaker. Add equal volume AcN and incubate 30 min at 37 C.
Pool supernatant with other one and add dithiothreitol to final concentration of 1 mM.
Reduce volume in speed-vac.
Zip-tip and concentrate in speed vac (not to dryness if possible).