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problem with purification of gst-tagged protein - (Jun/06/2012 )

hey guys!

i have a problem.

i expressed a protein in insect cells and now try to purify it. it is gst tagged and has a molecular weight of 120kDa.
everything works just fine but i am not able to elute the protein. if i check via westernblot it is still located on the glutathion-sepharose-beads.
i tried with an elution-buffer containing 40mM Glutathion.
do you have an advice for me?

thanks a lot!


did you use "reduced" glutathione? at pH 8?

the "book" says to load in pbs and elute with 50mM tris-hcl, 10mM reduced glutathione, pH 8.


yes i know, but neither worked. i really don't know why it doesn't work...