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?

Although, both enzymes have specific chemical inhibitors, the simplest way to quench the reaction (if they are to be run on SDS-PAGE) is to add SDS loading buffer and incubate for 3 min at 95°C. GluC and Trypsin are also inactivated by decreasing the reaction pH to 3. To inhibit them chemically, use TLCK or PMSF for Trypsin and 3,4- Dichloro-isocoumarin for GluC. However, the 3,4-Dichloro-isocoumarin reaction with GluC is partially reversible and can be incomplete. Therefore, SDS denaturation is best for gel analysis and the addition of 0.1% formic acid is recommended prior to MS/MS analysis. In general, chemical inhibitors are toxic, unstable and do not work as well.