My antibody does not work for IP, but works well fpr western? - antibofy not working for IP (Dec/06/2007 )
QUOTE (Telomerase @ Dec 6 2007, 01:17 PM)
I wonder if for co-ip, it shouldn't be monoclonal? It certainly wouldn't give unspecific background. With polyclonal, you can't be so sure if you co-ip the right thing, if the later step is spectrometry.
just because it is a polyclonal doesn't necessarily mean that it will give higher background. when it is produced there will be species of antibody that will recognize different parts of the antigen. the more unique the antigen, the less non-specific recognition sites. so, if you use a fragment of a protein that is unique in sequence as the antigenic agent, then your antibody will be more specific.
-mdfenko-
Are you doing IP for confirmation of interacting partners (found by other means, assuming), or for MS?
-Almasy-
QUOTE (chipping sparrow @ Dec 6 2007, 04:46 PM)
[ I run a western after the IP, there are some heavy chain visible, but much weaker compare to the IgG control.... 
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[/size]there are two important things for IP to work :
- the antibody must recognize the native form of the protein
- the antibody must be captured by the beads.
It seems here that the capture is not OK because you have much less heavy chain captured than the control. Did you incubate the same amount of antibodies than the control antibodies ?
-Missele-