Home Protocols Typical GluC In-Gel Digest Reaction Conditions (P8100)

Typical GluC In-Gel Digest Reaction Conditions (P8100)

  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, incubate for 5 minutes and invert the tube intermittently.
  3. Remove acetonitrile and repeat wash twice.
  4. Remove last acetonitrile wash and vacuum dry for 15 minutes.
  5. Add 100 µl of 1X GluC Reaction Buffer to rehydrate.
  6. Incubate 15-30 minutes at room temperature.
  7. Add GluC in a 1:10 or 1:20 protein substrate to GluC ratio (w/w).
    *Note: For example: Add 50-100 ng GluC to 1ug protein substrate
  8. Incubate at 37°C for 16 hrs.
  9. Desalt and purify peptides with C18 ZipTips prior to MS analysis.

Note: for reproducibility, all digestion reactions should be carried out for the same amount of time.