We suggest mixing the two enzyme buffers 1:1 GluC:AspN buffer. The Endo- proteinase GluC would prefer to have the E-E dipeptide present to stimulate activity and Endoproteinase AspN requires Zn, which is present in the AspN buffer. Still use a 1:20 (wt/wt) ratio of enzyme to substrate for each enzyme. There will be some reduction in the cleavage of your substrate protein since the two proteases will be chewing each other. It has been our experience that these conditions work well enough to get a high degree of digestion. You may need to adjust the individual amounts of each enzyme if you are missing a desired fragment, assuming that the reactions will be analyzed by some MS technique. If you prefer to do the reactions sequentially, do the Endoproteinase GluC reaction first as Endoproteinase AspN has difficulty digesting intact pro- teins. All digestions should be performed at 37°C .