Hi,
Thanks for replying to my post. My problem is (and I must phrase this delic
ately, as I am under a non-disclosure agreement...) that I have an antibody th
at recognises a common feature of several ER-resident proteins. I wish to conf
irm its continued activity (I re-generated it from an old stock of the cell li
ne in storage), and I attempted to do so by lysing some cells, hoping to see
numerous bands on a western of the lysate when probed using this antibody and
an alkaline phosphatase-linked secondary ab. This failed. So, I need to know a
way to enhance, ER-resident proteins in the mixture I apply to the gel...
My latest plan is to isolate ER-derived microsomes by sucrose gradient centrif
ugation, and hope that they retain a good level of their resident proteins.
I also have another cell line in culture that may be 'leaking' ER-proteins, an
d I'd like to use the same antibody to check the culture s/n for their presenc
e, but a simple blot of the s/n doesnt show anything either: I shall have to i
nvent some concentration protocol that also removes the fetal calf serum compo
nents as well....
It seems quite a hard pair of problems to me (I'm just starting my PhD) and th
ats why I turned to the net for help...
I hope you can give me a lead or two... 8)
