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Renaturing Far Western

Miguel Zamora mzamora at bcm.tmc.edu
Fri Jan 7 13:50:55 EST 2000


Bruno,

Take a look at these references.  I basically follow their protocol
with minor modifications,

Blanar,M.A. and Rutther,W.J.  Science,  (May 15, 1992) Vol. 256,
1014-1018
Vinson C.R. et al, Genes And Development 1988, 2, 801-806

Comments:
-  With my probes and target proteins, I could skip the
denaturation/renaturation step (although I include a washing step to
remove the SDS) but I get better signal if I include the
denat/renat. step.
-  Some people use PVDF instead of NC because of the binding
capacity and tighter binding.  However, in my case it gave me no
signal at all.  Maybe the binding of the target protein  to the PVDF
is too tight and does not allow for proper refolding of this protein
in particular.
- Keep in mind the source of the target and the probe proteins.  I
work with 2 human proteins, both of which bind to a third protein,
although to different sites (this is known from biochemical
studies).  For the probes, I use in vitro translation in RRL for
only one of them; express in bacteria for the other.  Of course,
there are several explanations for this.  My favorite is that the
first probe does not fold correctly in bacteria, but it does in
RRL;  as for the second probe, it is possible that it binds to the
rabbit homologous of the target protein and tight enough that it
does not comes off very efficiently. Let me know if you need the
protocol for making "clean" radioactive proteins in bacteria.


Let me know if you have any questions or need a more detailed
protocol.

Good luck!


Miguel



Bruno Cenni wrote:

> Hi all,
>
> I was looking for a detailed protocol on how to denature/renature
> proteins on membranes for Far Westerns. Does anyone know one?
>
> TIA
>
> Bruno
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