I have been working with GST tagged proteins for the last 4 years and after loading the cell lysate into the column I was washing it with 20-30 column volumes of PBS and sometimes my proteins were eluted very pure sometimes with a lot of impurities.

A few times I tried washing with NaCl gradient (0-400 mM) but later decided not to do that because it may denature the protein and some of my proteins may not refold.

What do you suggest for washing step?

Note: I don't have any UV detector to see the washouts/flowthroughs/elutions.

  • $\begingroup$ Use your plate reader to see the elution peak by collecting 100 ul or so as you are washing and eluting. You can also set up a Bradford-type colorimetric assay in a disposable microplate and add aliquots as you're washing and eluting to check for the presence of protein. $\endgroup$ – MattDMo Feb 8 '13 at 18:09
  • $\begingroup$ Bradford idea is really good actually, I will try that in my next purification. Thank you! $\endgroup$ – Engin Yapici Feb 8 '13 at 19:10
  • $\begingroup$ You're quite welcome. I did that before we got a little Biorad FPLC with a UV flow cell. If your lab does lots of purifications you might want to check it out - they're fairly inexpensive (as I recall) and you can just set the program up and walk away until it's done. $\endgroup$ – MattDMo Feb 8 '13 at 20:36

The standard when purifying GST with Glutathione agarose beads is to use STE buffer (10 mM Tris-HCl (pH 7-7.5), 150 mM NaCl and 1 mM EDTA) for washes and we have had no problem with this in our lab!


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