Protein A

[Scott Cowan]

Could anyone give advice on immunprecipitation methodolgy.
Presently I am attempting to ppt 32P labelled proteins
from various membrane fractions using polyclonals from
rabbit and ppt by protein A linked to agarose.
The main problems are high backgrounds in the pre-immune
controls. I`ve tried to decrease this using the method of
Platt et al (1986) Anal. Biochem 156, 126-135 but unfortunately
this also reduces the immune treatments to below detection
levels.
The main questions I would like to ask the net are
1) Can Protein A become phosphorylated,
2) Can IGG`s also become phosphorylated,
3) Is immunoprecipitation a viable technique to study 
in vitro 32P labelled proteins!

I would appreciate answers/advise to the above questions and also
any other problems I may have using immunoppt. I may have forgot to 
mention that the membrane fractions I use are obtained from pea 
epicotyl tips.
P.S 
sorry about the boring sig.