When I cut the GST-fusion protein bound to the column with glutathione sepharose 4B membrane with thrombin, the resulting elution contains a band at the correct size (72 kDa) followed by many smaller bands.
I also tried to cleave the eluted GST-fusion protein. In this case I see on SDS-page three bands corresponding to GST tag, thrombin and my protein.
I am using Glycerol,Triton, DTT, and the 25X complete cocktail to inhibit protease activity but with seemingly little success.
The lysis is done using a French press.
I am hoping to do immunization assay with my protein and need a very pure sample to do so.
If you have any suggestions I would be very appreciative.
I hope in your answer as soon as possible