In-gel Digestion Protocol for Endoproteinase LysC (P8109)

  1. Slice out the protein of interest from a band of an SDS-PAGE gel.
  2. Dehydrate the gel slice by adding 300 µl acetonitrile and mixing for 30 minutes at room temperature.
  3. Remove acetonitrile and vacuum dry 15 minutes.
  4. Add 100 µl of 10 mM DTT in 100 mM ammonium bicarbonate.
  5. Incubate 1 hour at 50°C.
  6. Remove DTT solution and add 100 µl of 50 mM Iodoacetamide.
  7. Incubate the gel piece for 45 minutes in the dark with intermittent gentle mixing.
  8. Wash the gel piece for 10 minutes with 100 µl of 100 mM ammonium bicarbonate.
  9. Dehydrate the gel slice with acetonitrile for ~15 minutes.
  10. Add 100 ng Endproteinase LysC in 100-300 µl 50 mM Tris-HCl, pH 8.5.
  11. Desalt and purify peptides with C18 ZipTips prior to MS analysis.