Home Protocols α-Lytic Protease In-gel Digestion Protocol (NEB #P8113)

α-Lytic Protease In-gel Digestion Protocol (NEB #P8113)

  1. 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.

  2. 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.

  3. Add 200 μl of acetonitrile, vortex mix for 30 seconds. Discard the supernatant.

  4. Vacuum dry for 5 minutes.

  5. Add 100 µl of fresh 25 mM DTT in 50 mM ammonium bicarbonate.

  6. Incubate for 20 minutes at 55°C.

  7. Remove DTT solution and add 100 µl of fresh 50 mM iodoacetamide in 50 mM ammonium bicarbonate.

  8. Incubate the gel pieces for 20 minutes at room temperature with intermittent gentle mixing. Minimize exposure of the reaction mixture to light.

  9. Remove iodoacetamide solution and wash the gel pieces with 400 µl of water by vortex mixing briefly. Discard supernatant and repeat once.

  10. 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.

  11. Add 200 μl of acetonitrile, vortex mix for 30 seconds. Discard the supernatant.

  12. Vacuum dry for 5 minutes.

  13. Add sufficient volume of 50 mM ammonium bicarbonate, pH 8.5 to just cover the gel slices.

  14. 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).

  15. Incubate at 50°C for 1 hour or 37°C for 16 hours.

  16. Collect the condensate from the tube walls by centrifuging at 12,000–16,000 × g for 10 seconds.

  17. Desalt and purify peptides with C18 ZipTips prior to MS analysis.