huangz at biowww.net wrote:
>In an immunoprecipitation-western blot I used rabbit polyclonal antibody and protein A agarose beads(protein A is covalently bind to beads) to pull down antigen complex. In following western blot I used rabbit polyclonal antibody as primary antibody and HRP-anti rabbit IgG as secondary. The result turns out to be many bands, including IgG bands. Is there a good way to do IP-W and get clean band? Any suggestion are appreciated.
I had the same problem. I was doing Western albeit without IP (rabbit
primary Ab, porcine anti-rabbit HRP-labelled secondary (DAKO Denmark),
ECL detection). The result was very similar. Seems the reason is not
IP but the antigen-primary Ab-secondary Ab interaction. The only
"clean" Western I have got was with biotinylated goat anti-rabbit
secondary antibody from Vector, streptavidin-AP coniugate and CSPD
chemiluminiscence (No affiliation to above mentioned companies).
Hope this helps,
Krzysztof Wasowicz, DVM, Ph. D.
Department of Animal Anatomy
Faculty of Veterinary Medicine
University of Warmia and Mazury
ul. Oczapowskiego 13 (Bldg. 105J)
Phone: +48-89-5233688, 5233733
Email: wasowicz at uwm.edu.pl