Typical Trypsin and GluC Co-Digest Reaction Conditions (P8100)

GluC works at maximal activity with a stimulating glutamic acid dipeptide in the reaction; calcium chloride reduces autolysis of Trypsin. Therefore, both of their unique buffers are used in this protocol at 0.5X concentration. Note that digestion of the proteases by each other will occur.

  1. Reconstitute Endoproteinase GluC in 1000 µl H2O
  2. Reconstitute Trypsin-ultra in 400 µl H2O
  3. Combine:
    3 µl H2O
    5 µl protein substrate (1 µg)
    5 µl 2X GluC Reaction Buffer
    5 µl 2X Trypsin-ultra Reaction Buffer
    1 µl 50 ng/µl Trypsin-ultra (NEB# P8101)
    1 µl 50 ng/µl Endoproteinase GluC (NEB# P8100 )
  4. Incubate at 37°C for 2-5 hrs.
  5. Immediately analyze by MS, MS/MS or store at -20 °C.