Two Hybrid Problem
DEZWAAN_T at a1.mscf.upenn.edu
Sat Apr 29 13:32:09 EST 1995
In article <hindges-280495162309 at 22.214.171.124>, hindges at vetbio.unizh.ch
(Robert Hindges) wrote:
> I am using pGBT9 for producing my protein as a fusion with the GAL4 BD. But
> I can't detect any fusionprotein in westerns loading about 40ug of protein
> extract on the gel. I sequenced of course the construct to proove in frame
> cloning. Any suggestions of more experienced user of this system would be
> appreciated. Thank you.
I'm actually not using this system but in my experience creating LexA DB
fusions three things come to mind:
1. What is the quality of your primary antibody that you are using for
the western blot? I also generated a fusion that sequenced in frame but I
couldn"t see on a western (even though I could see the LexA only positive
control). When I repeated the experiment with fresh antibody I could see
my fusion protein.
2. How big is the polypeptide that you are fusing to the GAL4 BD? When I
fused a 110kD protein to the LexA DB I could see it on a western but the
signal was greatly reduced when compared with a truncated version of the
construct which carried only the C-terminal half of the protein (size of
the fusion product = 80kD)
3. How do make your yeast extracts? I find that if I grind my yeast up
with glass beads on ice, etc. that I don't get as much protein recovered
as when I simply take 1 ml midlog cells (OD600=0.5), spin them down for 3
min. top speed, add 50 ul 2X Laemmli and immediately put at -70 c. I then
load about 25 ul on a gel.
Hope this helps.
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