purifying antibody in small volume

[Michael Sullivan]

There is a lab manual on antibodies by Edward Harlow published by  
Cold Spring Harbor that would probably have useful information about  
this.

Two approaches that come to mind for me are ammonium sulfate  
precipitation and ultrafiltration. If you have background issues that  
require preclearing, maybe a good approach would be to do AS  
precipitation on your serum, then do your preclearing procedure on  
the partially purified antibody preparation.

Hope this helps.

Mike


On Apr 30, 2007, at 5:58 PM, fabianh from umich.edu wrote:

> 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
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)