In article <20021213023520.1362.qmail at ww02.hostica.com>, ruben_martherus at yahoo.com wrote:
>I've expressed a GST-fusion protein (original size ~24 kDa). The protein is
> expressed and shows on SDS-PAGE a band of correct size. However my
> purification of the protein (using Glutathione-Agarose beads, Sigma according
> to manufacturer) is not working. I've tried things like, changing buffers, pH,
> incubation time etc. The control (GST only) works ok, and also another fusion
> protein has no problems. The protein fused to GST is highly negatively charged
> at the pH at which the experiments are done. Does somebody have any
> suggestions?
What exactly does "is not working" mean? Generally, if the problem is ionic
interactions - in your case that would be a repulsion - raising NaCl to something
in between 0.5-1.0 M frequently helps.
DK
