α-Lytic Protease In-gel Digestion Protocol (NEB #P8113)
- Excise the band from an SDS-PAGE gel corresponding to the protein of interest. Cut the gel slice into three 1mm pieces and transfer them to a 1.5 ml microcentrifuge tube.
- Dehydrate the gel slices for 5 minutes in 200 μl of acetonitrile:50 mM ammonium bicarbonate (1:1 v/v) with intermittent vortex mixing. Discard supernatant.
- Add 200 μl of acetonitrile, vortex mix for 30 seconds. Discard the supernatant.
- Vacuum dry for 5 minutes.
- Add 100 µl of fresh 25 mM DTT in 50 mM ammonium bicarbonate.
- Incubate for 20 minutes at 55°C.
- Remove DTT solution and add 100 µl of fresh 50 mM iodoacetamide in 50 mM ammonium bicarbonate.
- Incubate the gel pieces for 20 minutes at room temperature with intermittent gentle mixing. Minimize exposure of the reaction mixture to light.
- Remove iodoacetamide solution and wash the gel pieces with 400 µl of water by vortex mixing briefly. Discard supernatant and repeat once.
- Dehydrate the gel slices for 5 minutes in 200 μl of acetonitrile : 50mM ammonium bicarbonate (1:1 v/v) with intermittent vortex mixing. Discard supernatant.
- Add 200 μl of acetonitrile, vortex mix for 30 seconds. Discard the supernatant.
- Vacuum dry for 5 minutes.
- Add sufficient volume of 50 mM ammonium bicarbonate, pH 8.5 to just cover the gel slices.
- Add 100 ng of α-Lytic Protease (dilute 0.4 mg/ml α-Lytic Protease to 0.1 mg/ml in 10 mM sodium acetate, pH 5.0, then add 1 µl to the gel slices).
- Incubate at 50°C for 1 hour or 37°C for 16 hours.
- Collect the condensate from the tube walls by centrifuging at 12,000–16,000 × g for 10 seconds.
- Desalt and purify peptides with C18 ZipTips prior to MS analysis.
