Dennis J. Templeton
djt2 at po.CWRU.Edu
Fri Oct 2 16:16:09 EST 1992
In a previous article, skspoidn at susssys1.reading.ac.uk () says:
>I am trying to Immune precipitate a cellular protein
>(CD4) with Mabs using protein A and S35 metabolically
>labelled cells, and finding that the immune reaction is
> not occuring, although antibodies are being precipitated.
> Is there some aspect of the immune reaction and susequent
> precipitation which is critical in this procedure?
You should be aware that some, even most, monoclonals don't bind directly
to PrA, I'd cite the classes here but I'd probably mix them up. We usually
test a new mAb empirically by binding some to PrASepharose and eluting and
staining for the IgG on a PAGE.
If your mAb doesn't bind to PrA directly, not all is lost, as you can add
rabbit anti-mouse IgG to sandwich the mAb to the PrA with the rabbit IgG
that binds pretty efficiently. In my experience, the Rabbit anti-mouse
serum doesn't affect the quality of the IP's very much.
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