May a CARRIER-antibody compete between them for binding to protein G-agarose? - Immunoprecipitation problem (Sep/14/2006 )

Hi, all

I want to perform an immunoprecipitation experiment. To do it, I have an antibody which is accompanied by gelatin as a carrier. On the other hand, I have proteinG-agarose. I fear that gelatin compete for binding to proteinG-agarose, finally reducing the amount of Ab-proteinG. So, should I use a carrier-free antibody? or maybe a pre-blocked proteinG?
I wait for your answers.

Thanks in advance

I have no experience with gelatin-conjugated antibodies but I had a look at this paper http://www.sciencedirect.com/science?_ob=M...=/sdarticle.pdf where you can see a schematic representation of the gelatin-antibody structure. The gelatin molecule looks quite big and I have the impression you could have problems of steric hindrance between the gelatin and the protein A (the gelatin is very close to where the protein A binds).
Maybe you could incubate the Ab with prot A and then have a look at how much binds the preoteinA and how much stays in solution, comparing to a uncoupled Ab.
i hope it helps.
Good luck!

If I understand well, it's not a gelatin-conugated Ab.

I don't think it will interfere. I already used fish gelatin to block the membrane for western-blot, but did'nt interfere with the antibody binding to the antigen.
I'm quite sure that the specific interaction between the protein A and the Ab will be stronger than the non specific interaction between gelatin and protein A or the Ab.

(Carrier free antibody are offered to people who you want to label the antibody.)

Thank you for your answers. At least I have one thing clear, my ab is not conjugated to gelatin, which is only a preservative adedd to the liquid where the ab is included. In the next few days I will performance the IP. I will tell you the results and conclussions.
Again, thank you, missele and dnafactory.

You were right. Apparently, there were no problem.

Thanks

You were right. Apparently, there were no problem.

Thanks

You were right. Apparently, there were no problem.

Thanks

You were right. Apparently, there were no problem.

Thanks

You were right. Apparently, there were no problem.

Thanks