On Mon, 21 Dec 1992 Mark.OLeary at newcastle.ac.uk wrote:
> Date: Mon, 21 Dec 92 10:39:51 GMT
> From:Mark.OLeary at newcastle.ac.uk> To: GARBRECHT <fgarbrec at post.its.mcw.edu>
> Cc: immunology at net.bio.net> Subject: Re: ER - resident proteins
>> 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)
As far as concentrating your ER proteins goes, your plan sounds reasonable
to me. In terms of seeing if your antibody has activity, well, thats another
problem. Several things come to mind; first, if the hybridoma is old but
viable, it probably would benefit from re-cloning as hybridomas tend to spit
out chromosomes along the way. (Obviously you need a screening method to
do the recloning.) Also, check the hybridoma for mycoplasma contamination;
this is a problem in lots of labs, and can lead to poor antibody secretion
in otherwise viable cultures. It might be helpful to talk to the person who
originally did the fusion for details on their original screening protocol.
The best way to check your detection system for problems would be to run
a control antibody that you know has activity (of the same subclass) against
some other protein known to be expressed by your cell line (or tissue); you
can troubleshoot the assay from there. Make sure all of your antibody
reagents etc are reasonably fresh and have been stored appropriately.
Good luck,
Fred Garbrecht
Medical College of Wisconsin
fgarbrec at post.its.mcw.edu
