Two hybrid Q.
bernard at elsie.nci.nih.gov
Tue Mar 14 20:55:13 EST 1995
In article <Pine.SOL.3.91.950314131706.24432E-100000 at corona>, Rafael Maldonado <rafael at corona> writes:
> Hi folks!
> I'm trying the two hybrid method for detecting interacting proteins in yeast.
> I have seen that the proteins to be tested are disposed in the C-terminal
> of the gal4 protein domains (both). I mean, the fusions are always in the
> N*gal4domain-yourfavoriteprotein*C direction.
> Because I'm interested in the interaction with the N-terminal end of my
> protein, can I use the fusion in the other way, N*Myprotein-gal4domain*C?
> Have anyone tried that? Do you think it would work?
> Rafael at genetics.med.utah.edu
I am interested in this as well. I asked the Clontech representative
about them providing N and C terminal fusion vectors when they gave a
symposium locally last year. I was told that they were working on this
and were also intending to provide vectors with multi-cloning sites in
all three reading frames. However, I have yet to hear from them again.
If anyone was to build their own vectors that would allow such choices
I would be very interested to hear from them. I think accepting the
current kit as it stands is a little short sighted as although the
termini of proteins may often not be functionally important/important
in protein-protein interactions this is not always the case and all
four combinations of N and C fusion should really be tested for each
Just my dha/ phingin worth....
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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