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.