have you tried preincubating your lysate with GST alone? this usually
helps reduce the background.
thomas
On Thu, 18 Jul 1996, Pavel Sova wrote:
>> Hi everyone,
> I have a problem with studying interactions of protein of interest (HIV-1
> Vif) fused with GST, with other proteins. When I load GST-Vif onto
> glutathione-agarose (or sepharose) beads and use this as a binding matrix,
> I get basically the same proteins binding to control beads (loaded with
> GST-unrelated proteins), but not to beads which were not loaded with any
> protein.
>> As a binding and washing buffer, I use:
>> 20 mM Tris.Cl, pH 7.5
> 100 mM KCl
> 2 mM CaCl2
> 2 mM MgCl2
> 5 mM DTT
> 5% glycerol
> 0.5% NP-40
>> Usually, after incubation with lysate of cells or protein I am trying to
> see whether it interacts with GST-Vif, I wash beads (vol. of 25 ul) three
> to five times with 1 ml of the same buffer. I tried to use different
> buffers, with ionic strength and content of detergents similar to the
> above, but without much success to get specific binding with only GST-Vif,
> but not other GST-fusions loaded on beads.
>> Did anyone of you run into same problem and how did you solve this??
>> TIA for any input
> Pavel
> -----------------------------------------------------
> Pavel Sova e-mail:
> Molecular Virology Laboratory ps44 at columbia.edu> Columbia University
> New York
> -----------------------------------------------------
>>>>>
Thomas Seng-Lai Tan ...................................
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