Problem with MBP and GST tag purification - (Aug/23/2010 )
I have problem with the expression of my gene. I clone 3.2 kb gene into pMAL and pGEX and express in Rosetta (DE3. The expression is very low. Also I have problem with purification. I get very very low purity. Actually, after past soluble protein through the beads and wash until I'm quite sure that their no unbound protein left. The elude fraction still get almost every bands in it, not only my target protein. It happen with both vectors. I also tried western blot and found that it react to every band in elution fraction too.
I already tried to add glutathione in my washing buffer in a low concentration but result was still the same.
EXCESSIVE SONICATION DISRUPTS THE GST PROETIN AFFECTING ITS BINDING TO THE RESIN DECREASE SONICATION