This procedure should be included when maximum of protein coverage is required, or when digesting a band from a 1-D gel. Depending on your procedure for 2-D gel electrophoresis, proteins may be already reduced and alkylated, so this procedure may be omitted.
- Add enough 10 mM DTT solution (~50 µl) to cover the gel pieces, reduce for 1 hour at 56° C
- Cool to room temperature and replace teh DTT solution with roughly the same volume of 55 mM iodoacetamide solution. Incubate for 45 minutes at room temperature in the dark with occassional vortexing.
- Wash gel pieces (rehydrate) with ~100 µl of 25 mM ammonium bicarbonate, pH 8, for 10 minutes while vortexing and dehydrate with ~100 µl of 25 mM ammonium bicarbonate/50% acetonitrile. Repeat rehydration and dehydration.
- Remove the liquid phase and dry gel pieces in vacuum centrifuge