GST-activity, binding buffer

[Noam Harel]

you at somehost.somedomain (Maor Bar-Peled) wrote:
>
> Dear Netters; I was wondering if someone has tried systematically to check whether 
> the GST expressed in E.Coli (using the pGEX system) binds the GSH-column
> under different buffers, detergents, salts etc conditions?. 
>  i.e Does extraction in Hepes or 
> tris buffer allow similar binding of the GST to the column compared with PBS? 
>              Does the GST activity remain the same in Hepes or tris buffers as
> in PBS?
> Thanks in advanced, Maor Bar-Peled.

Maor,

	There are almost as many buffers and conditions used in GST
experiments as there are labs performing them. Pretty much any 
non-denaturing buffer will do, including PBS, Tris and Hepes.
GST alone will bind equally and strongly to glutathione beads in any
of these buffers at pH 7-8, NaCl 100-500mM . But GST-fusion proteins
are more variable, and you need to do a titration of salt and/or
detergent (NP-40, start at 0.5%) to see the effects on your protein.
The effect of conditions is much more important when looking for
interactions of your GST-fusion protein with other, non-GST proteins,
rather than the interaction of the GST-fusion with glutathione beads.
There are numerous papers with details on this technique. Read a few
to get more of a background. Good Luck!

Noam