purification of a basic protein - problem in elution (Mar/10/2009 )

I am using the PGEX system to purify a basic protein of PI 9. I am using a denaturing 2M urea based protocol that i came across in a paper. The lysis & wash buffer have Tris, EDTA, B-ME and 2M urea. The protein seems to be binding the column but when I try to elute it with PBS having 10mM glutathione + 2M urea nothing comes off. Can someone suggest some variations I could try to recover it from the column. Will changing PH conditions help?

First of all, I'd try more glutathione, even up to 100 mM.

How do you know so clearly that your protein has been bound to the resin?. Maybe you have aggregation and you need to increase the urea till 6 or 8 M. 10 mM gluthation is the suggested concentration for GST elution. At least in my hands, the affinity of a recombinant protein fused to GST to its resine usually is very low, and a binding situation which is not eliminated with 10 mM of gluthation is not very common.

Hey

I also face this problem of protein not binding to beads under denatured conditions. I refold the whole lysate and then bind.

Alternatively, give a lot of washes with 1M urea and get rid of the proteins soluble in 1M urea. Now give repeated 2M washes and run the fractions on a SDS-PAGE gel. Refold the ones which are relatively pure, bind it to an anion exchange column and purify or change the buffer.

best
TC

In reply to Swanny - apparently increasing glutathione to 100mM would also mean increasing the salt concentration. I have read that people have gone upto using 500mM NaCl with 50mN glutathione. any suggestions if you have tried 100mN GSH.

In reply to paramyosin - using such strong denaturants like 8M urea or guanidine might denature the GST or column to an extent that it might even affect binding. maybe using 8M urea for just the elution can work.also i was wondering that since the binding of the GST tagged protein to the glutathione column is based on a covalent disulfide linkage would it worth including a reducing agent like mercaptoethanol in the elution buffer. any suggestions?

thank you