epitope tagging for ovary detection

[sea]

In article <4gofqg$49r at nntp3.u.washington.edu>, dgilles at u.washington.edu
(Doreen Gillespie) wrote:

> I am looking into possible epitopes to use for tagging a protein 
> expressed in the germline.  Does anyone have suggestions, experience, 
> epitopes to avoid?  My understanding is that myc tags have been extremely 
> variable, due in part to poor antibodies.  Has anyone tried the 
> hemaglutinin epitope?  One of my key concerns is high background--the 
> 9E10 myc antibody isn't fantastic, and I don't know about the 
> hemaglutinin antibody (BAbCO).
> 
> Thanks for any thoughts,
> Doreen Gillespie

Dear Doreen

I've used the FLAG epitope system with a C-terminally tagged protein. In
Drosophila there are problems with the anti-FLAG monoclonal antibody that
you have to use for a C-terminal FLAG, the M2 monoclonal antibody. The M2
antibody cross reacts with a couple of proteins in Drosophila embryos and
the only tissue I have found so far where the M2 antibody reacts cleanly
with the ectopically expressed and FLAGged protein is the adult brain.
There is a new anti-FLAG monoclonal antibody released by Kodak, the M5
clone which is supposed to react with an N-terminal FLAG epitope. The M5
clone is reputed to have minimal cross reactivity in Drosophila. The man
to speak to is Bill Brizzard at Kodak. Unfortunately I dont have his
e.mail address to hand but I think it may be in the Kodak catalogue.

Hope this helps

Sean Sweeney

p.s. I have no connection with Kodak whatsoever and all the usual
disclaimers apply.