Dissolve 500 units of thrombin into 500 microliters of cold phosphate buffered saline solution. Swirl gently to make the protease dissolve. This makes a thrombin solution of 1 U/microliter. If you are using another protease, follow the instructions specified in your GST manual.
Mix 80 units – 80 microliters – of thrombin into 920 microliters of PBS buffer for every milliliter of glutathione sepharose used in your column. For example, if you used 2 milliliters of glutathione sepharose to make your column, use 160 microliters of thrombin and 1,840 microliters of PBS. If you are using another protease, follow the directions specified in the manual when making your protease mixture.
Apply the thrombin mixture into the glutathione column you have previously applied your GST tagged protein. The GST tag binds to the glutathione, holding your protein within the column. You can also remove the GST tag after you have eluted your protein from the column by mixing your protein with the thrmobin mixture in a tube.
Seal the column and incubate at room temperature for 2 to 16 hours. Some proteases can be incubated in 4 degrees Celsius. Follow the specific directions in the GST manual.
Wash the column with 3 column volumes of PBS buffer. Collect the flow-through into a few separate tubes and analyze them. The flow-through contains your protein as well as thrombin, while the GST tag remains bound into the column. If you remove the GST tag in a tube, your free protein will be mixed in with the GST tag and the thrombin.