immune precipitation

skspoidn at susssys1.reading.ac.uk skspoidn at susssys1.reading.ac.uk
Wed Oct 7 03:29:20 EST 1992


(others have asked for this so...)
The only reply that I have got so far, which I have checked and unfortunately 
does not apply in my case, is that not all Abs bind protein A, and that there 
is a level of affinity at the Ig subclass level. It is best that the system
 be optomized empirically with the ab of interest.

The paper I originally got the protocol from (Borchelt et al., 1992: 
J Biol Chem v267 pp16188-16199) also suggests that the methanol 
precipitated proteins be resuspended in 3M guanidine thiocyanate,
 20mM Tris pH 7.5 for 10 min, followed by reprecipitation with 10
 vol methanol. This extra denaturation step apparently allows for 
a more quantitative dispersion of protein into DLPC. Note that this
 may only apply to the protein that they were working with (Prp).

This post was actually for a colleague, and he assures me
 that subclass affinity is not a problem in this case, so 
I repost my original question, being are there any (other)
 critical factors which influence immunoprecipitation?

Thanks in advance,

Mike Poidinger
Dept of Microbiology
University of Reading



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