| Migs said: |
| I am purifying a GST tagged protein with contaminants!. How do I get rid of them. I need active protein, what conditions I can change. The protein is found in inclusion bodies and I extract it with sarkosyl and Triton X 100. I have tried 500mM salt conc. in wash buffer, extensive washing, addition of glycerol and low resin bed volume to allow competition for binding. What more I can do to get rid of the non specific bands. |
Migs - I assume you mean that after you run the glutathione-sepharose column you have contaminants? What do you know about the contaminants - could they be fragments of your protein that also have GST on them, and are therefore copurifying? If so, it won't matter how much you wash, they'll still copurify with your desired protein.
If this is the case, an orthogonal step (e.g. size exclusion, ion-exchange) would be the way to go. Typical resins would be Q or SP, depending on the pI and the pH tolerance of your protein.
PW