Typical GluC In-Gel Digest Reaction Conditions (P8100)
- Slice out the protein of interest from a band of an SDS-PAGE gel.
- Dehydrate the gel slice by adding 300 µl acetonitrile, incubate for 5 minutes and invert the tube intermittently.
- Remove acetonitrile and repeat wash twice.
- Remove last acetonitrile wash and vacuum dry for 15 minutes.
- Add 100 µl of 1X GluC Reaction Buffer to rehydrate.
- Incubate 15-30 minutes at room temperature.
- 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
- Incubate at 37°C for 16 hrs.
- 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.