New England Biolabs, P8100S, Endoproteinase GluC

Related Categories Proteases,, Proteome Analysis Applications Glycomics and glycoproteomics,, Biotherapeutics and Antibody Analysis,, Protein Digestion Specification Reaction Conditions 1X GluC Reaction Buffer Incubate at 37°C 1X GluC Reaction Buffer 50 mM Tris-HCl 0.5 mM Glu-Glu (pH 8 @ 25°C) Usage Concentration 100 ng/μl Molecular Weight Theoretical: 29849 daltons FAQ Q: What is the stability/compatibility of Endoproteinase GluC with regards to presence of DTT, urea, concentrations of methanol, acetonitrile, nitrosoguanidine, thiourea and glycerol? A: GluC has no cysteines and therefore is not affected by DTT. GluC has reduced activity in the presence of glycerol. 4M urea will denature GluC. Any denaturing should be done with a brief heat treatment, such as 95°C for 1 min. If 4M urea is necessary, use it to denature the substrate protein and then dilute the reaction 10-100 fold before digestion with GluC. Low levels of nitrosoguanidine or thiourea may be tolerated by GluC; however, both are chaotropic agents and may have to be tested empirically. GluC is compatible with 10-20% methanol or acetonitrile; however, acetonitrile is recommended over methanol. When using organic solutions with GluC, results may vary depending on the protein substrate. Q: 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? A: 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.