Western blot problem, need suggestions

Peter Dudek peterd at primeachip.com
Tue Jul 10 08:33:40 EST 2001


Oddie,

First, we need to know a little bit more about your experiment.

(1) did you do a coomassie stain before transferring? what was the result?
(2) did you do a ponceau stain before blotting? what was the result?
(3) are you trying to detect endogenously expressed Cry toxin receptor? or
did you overexpress it? if overexpressed, is it tagged (His, GFP, FLAG, myc,
GST etc?)
(4) check to see that your primary antibody came from rabbit (otherwise your
anti-rabbit secondary would be useless)
(5) did you have a positive control to make sure the AP reaction worked?
(6) did you try with an HRP secondary?

After reading your original post in more detail, I realize I was a little
hasty in my response... so, just that I'm clear, you are trying to detect a
receptor on a membrane by adding the ligand, then immunoblotting against the
ligand?? Has this method been previously published, because I would imagine
that it wouldn't work. Your receptor is denatured on your membrane (assuming
you used an SDS-PAGE gel), and so I wouldn't expect that its natural ligand
would stick to it all. Anyways, we need some more details before we can help
you.

cheers,
Peter



"oddie" <teerachild at yahoo.com> wrote in message
news:20010710114049.6639.qmail at ww02.jatek.com...
> sorry guys.
> I used AP not HRP.
>
>
> <http://www.biowww.net/forum/read.php?f=1&i=2881&t=2880>
>





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