immunoprecipitation problem - (Feb/08/2006 )
Another option is to do the westernblot with an antibody made in another specie different from the specie in which the antibody for the immunoprecipitation is made. In other words, if you make the IP with a polyclonal antibody (usually rabbit), then use a monoclonal antibody for the westernblot detection (usually mouse). The monoclonal antibody doesn't detect the rabbit immunoglobulins.
Hi Nagroc,
I have done what you suggested. IP protein (A) with rabbit polyclonal antibody, and detect protein (
with mouse monoclonal antibody. However, the heavy chain band still appear. I think it is because of the cross-reactivity (althogh it is low) of the secondary antibody to the IP antibody. It is the case. How can we deal with it? 
Hi, My first idea to deal with it, is to make a higher dilution of the secondary antibody, and play with the exposition time in order to get an image in which appears the desired protein and doesnt appear the immunoglobulins.
a better option would be to use protein A-HRP to detect your monoclonal antibody instead of the anti-mouse, because protein A will only recognize the native form of the monoclonal antibody and not the denatured antibody used for IP.
I have some problem with immunoprecipitation.... I'm styding Src kinases family, which has 60 KDa of molecular weight... the problem is: when I reveal immunoprecipitates with anti-Src antibody, a enormous band appears (IgG) which doesnt let me watch Src band because of both, src and IgG bands, are very close...
What can I do to avoid this fact???
Thank you!!!
had the same problem with Src IP.. and also rabbit Ab i used for detection crossreacted with mouse heavy chain of Ab i used to IP.
so: i did IP with mouse Ab, loaded 12% gel - Src is 60, so i let the 50kDa marker band to run out, and blotted with rabbit Ab - and it was fine.
Hi!!
I have some problem with immunoprecipitation.... I'm styding Src kinases family, which has 60 KDa of molecular weight... the problem is: when I reveal immunoprecipitates with anti-Src antibody, a enormous band appears (IgG) which doesnt let me watch Src band because of both, src and IgG bands, are very close...
What can I do to avoid this fact???
Thank you!!!
had the same problem with Src IP.. and also rabbit Ab i used for detection crossreacted with mouse heavy chain of Ab i used to IP.
so: i did IP with mouse Ab, loaded 12% gel - Src is 60, so i let the 50kDa marker band to run out, and blotted with rabbit Ab - and it was fine.
I used HRP conjugated protein A from GE health and it worked very well. Protein A will only bind to primary antibody. I think the sensitivity is a little low compare to regular 2nd AB.
Many thanks for all.
Hello IPers:
The problem you are describing with respect to the heavy chain are not uncommon.
For this reason, Santa Cruz Biotechnology has developed an Exactacruz Kit which is specifically designed to eliminate detection of the heavy chain on a Western blot following IP.
There are 6 systems total; three homologous systems where a mouse IP/mouse WB antibody is used, or a rabbit/rabbit or a goat/goat is used; and three heterologous systems using combinations of mouse, rabbit and goat primaries for IP or WB.
Check out the website at www.scbt.com and click on "What's New" : Exactacruz for more information.
Also, feel free to contact our Technical Service if you would like more information. These kits were formulated to deal with the exact problems that you have been discussing in this topic!
Our phone number for Technical Service is 1-800-457-3801 ext. 2, and our Technical Service email is scbt@scbt.com.
Good luck with your research!