Immunoprecipitation

Santosh santi at MCBL.IISC.ERNET.IN
Sun Mar 8 00:20:15 EST 1998


Hi Folks!!
 I am trying to purify a mycobacterial protein by immunoprecipitation 
using polyclonal antibody raised against this crude electroeluted ( from 
10% SDS-PAGE gel) protein, in rabbits. But the problem is that there are 
other proteins of exactly same molecular weight where my protein of 
interest is located. I have confirmed this by 2DElectrophoresis. Can 
anybody help me in solving this problem of getting my protein in pure 
form and getting rid of the other comtaminating proteins? 2DE shows that 
the pI of my protein of interest and also two other contaminating 
proteins lies in the acidic region (though i haven't been able to find 
the exact values of these, but the spots i get are not too far off 
from each other i.e. they are a bit close together).
 Also after immunoprecipitation, i would like to get back the unbound 
antibody. So how do i separate the immunoprecipitate (antigen-antibody 
complex) from the unbound antibody so that i get the pure unbound antibdy 
for reuse? 
 If anybody can help me find answers to these problems please email me at 
the following email address - 
         santi at mcbl.iisc.ernet.in
Thanx.
- Santosh



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