purifying antibody in small volume

fabianh from umich.edu via methods%40net.bio.net (by fabianh from umich.edu)
Mon Apr 30 17:58:39 EST 2007


Hi,

Does anyone know of a way to purify antibodies resulting in small (and 
concentrated)volumes?

I got an antibody which is not very strong. I have to use it in a 1:800 
dilution at the most to detect anything on my blots. the problem is 
that I also detect several other proteins. I tried to purify it by 
incubating with e.coli celllysate of a nullstrain for my protein and it 
worked quite well (only one band left) but now it is even weaker as 
this protocol uses SDS and a total volume of 10ml for 100ul of 
antibody. So my purified antibody is diluted 1:100 already leaving me 
with a 1:8 dilution (which then needed a 45min exposure of the film to 
detect weak bands)! And i would need a really strong signal for my 
purposes.
Thanx a lot for any help

Fabian

Bardwell Lab
University of Michigan



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