Protocol using Endoproteinase AspN (NEB #P8104)
Endoproteinase AspN (NEB #P8104) is typically resuspended to 0.1 µg/µl in highly pure water. Endoproteinase AspN does not digest proteins greater than 5 kDa efficiently. In this case, a double-digest is recommended. Similar to trypsin or GluC digests, substrate protein to protease mass-to-mass ratios are recommended in the range of 20-50:1.
Protocol 1. Endoproteinase AspN Digestion
- 1 µl (2 µg) substrate protein
- 1 µl (0.1 ug) Endoproteinase AspN
- 10 µl 2X AspN Reaction Buffer
- 8 µl H2O
Incubate 2-18 hrs at 37 °C
Protocol 2. Double Digest with Endoproteinases AspN and Trypsin or GluC
Simultaneous digestions may be carried out with Endoproteinase AspN and another protease. A rule of thumb is to use 0.5X buffer final concentration for each proteases unique buffer. For the example here, the reaction will contain both zinc for Endoproteinase AspN and Glu-Glu dipeptide for Endoproteinase GluC reaction; the mass-to-mass ratios are 20:1 for substrate to each individual substrate. If doing sequential digests, we recommend using Endoproteinase AspN second in the workflow.
- 1 µl (2 µg) substrate protein
- 1 µl (0.1 µg) Endoproteinase AspN
- 1 µl (0.1 µg) Endoproteinase GluC
- 5 µl 2X AspN Reaction Buffer
- 5 µl 2X GluC Reaction Buffer
- 7 µl H2O
Incubate 2-18 hrs at 37 °C
