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
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Pavel Sova e-mail:
Molecular Virology Laboratory ps44 at columbia.edu
Columbia University
New York
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