I am doing a protein purification of GST from E.coli cell lysate. I need to purify this protein under naturing conditions using Glutathione Agarose beads. I have no more glutathione. Does anyone know how I can elute my protein without glutathione? And also without denaturing my protein?
It's possible to elute with a protease, provided there's a cleavage site between the tag and your protein. Then you'd have to get rid of the protease, though.
Depending on the GST vector, there is often a thrombin or precision protease site located between the GST and the fusion protein, so as Andrea says, you can use this to cleave your product (although you'll lose the GST tag)
I would say the best is to buy some reduced GSH in order to keep your enzyme reasonably active !
However, you may try small concentration of another mercaptan ( thiol group-containing) such as DTT or cysteine but I am not sure of concentration needed and that you'll keep your enzyme active? Also reactivity of your GST cysteines is not to be excluded.
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