immune precipitation

Basavaraju Shankarappa bsh at MED.PITT.EDU
Mon Oct 5 08:42:11 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?
> >
> 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.
> dennis
	One suggestion to overcome these problems.  I had used this procedure
with some success dut did not need to explore in detail.  
Can we not use the say anti-mouse immunoglobulins that are bound to a solid-
phase.  Since these are polyclonal, I think it should be fairly easy for them
to bind as opposed to the limited binding epitope of ProteinA.  I know two
suppliers one is Kirkegaard and Perry Lab at Gaithersburg Md and the other is
Pierce (Call 1-800-555-1212) for both Toll free numbers.  
	If somebody has tried this method, can you please add your comments?
Raj Shankarappa
Univ of Pittsburgh.




More information about the Methods mailing list