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?
1. try changing ionic strength
2. try adding detergents/organics
3. you might be screwed - on occasion, the fusion protein will cover the GST
active site so much that the tag will not work no matter what. solutions
range from introducing long flexible linkers (might not work due to
potential for preferential association) to re-cloning with a different tag
or in a tag sandwich
4. minuscule probability of a blooper or mutation - can you make sure that
your GST tag is indeed GST - mass spec the partially purified fusion for
instance.
A.G.E.
