GST-fusion interaction--high background??

Seng-Lai Tan sltan at u.washington.edu
Thu Jul 18 19:04:37 EST 1996


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               ...................................
Dept. of Microbiology		  : Definition of politics          :
Box 357242			  :  poly = many or excessive       :
University of Washington	  :  ticks = bloodsucking creatures :
Seattle, WA 98195 		  : Any questions? :)               :
                                  :.................................:
Phone:  206-543-6700
FAX: 	206-543-8296




More information about the Proteins mailing list