Thrombin inactivation - (Aug/29/2013 )
I am using thrombin to remove a GST tag from my fusion protein. I am doing a time-course to check how long to incubate my protein with thrombin. I decided to add samples straight to my sample loading buffer at the appropriate time point and I will load the gel the next day. The final concentration of beta-mercaptoethanol and SDS in the sample buffer will be 5% (710mM) beta-ME and 10% SDS. I have read that 10mM beta-ME and 0.01% SDS do not inactivate thrombin. Does anyone know if 710mM beta-ME and 10% SDS will completely inactivate the thrombin?
Are you sure your final concentrations of BME and SDS are 5% and 10%, respectively? I suspect those are the concentrations in your 5x sample buffer, which gets diluted 5-fold with your protein so that your final BME and SDS are 1% and 2%. Regardless, thrombin will likely be denatured in those conditions...to be sure if you boil your sample at 95C for 5 min you will certainly inactive it. Stick your sample in the -20C after boiling and run at your convenience.
Also, TLCK will inactivate thrombin irreversibly. You can buy it at Sigma or most any chemical vendor. Add it to your sample prior to adding your SDS sample buffer.