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  • FAQ: How does one carry out limited proteolysis experiments with Trypsin and Endoproteinase Glu-C? Which chemicals can be used to quench the reactions at given time points to monitor proteolysis progress via SDS- PAGE?

    While both enzymes have specific inhibitors we suggest the simplest way if they are to be run on SDS-PAGE is to just quench them with SDS loading buffer and heat. Both are also inactivated by taking the pH to 3. To inhibit them chemically we suggest TLCK or PMSF for Trypsin and 3,4- Dichloro-isocoumarin for Endoproteinase GluC. The 3,4-Dichloro- isocoumarin reaction with Endoproteinase GluC is somewhat reversable and can be incomplete. SDS denaturation is probably best for gel analysis and formic acid addition to 0.1% for MS/MS analysis. In general the inhibitors are toxic, unstable and not worth the trouble.