GST-fusion interaction--high background??

Pavel Sova ps44 at columbia.edu
Thu Jul 18 12:47:32 EST 1996


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                            
 -----------------------------------------------------






More information about the Proteins mailing list