Hi everyone, This is although an off topic question but I would certainly seek expert advices on the following query:
I have a GST-tagged protein that is purified in presence of the steroids. I carry out an in column digestion using thrombin. Digestion buffer compositin: 50mM Tris pH: 8.0, 150mM NaCl, 10% glycerol, 100micro Molar steroid, 0.15 n-octyl-beta glucoside, 1mM DTT, 3mM CaCl2, 12units thrombin/ml of protein Problem: Protein when purified in presence of steroid like testosterone gets around 85% digested on incubating at 4 degree C for 24 hours with the above specified thrombin quantity. However, when the steroid is replaced by some plant steroids like phytoestrogen there is no GST digestion under the same conditions? Can someone provide some inputs for achieving the perfect GST-digestion? Whether a change in some of the constituents of the buffer or some other factor could be effective? Thanks in advance for any suggestions. Cheers Gauri
