I precipitated my protein with an antibody generated in a rabbit. For detection I use one that is generated in a mouse. When detecting I still observe the heavy and light chain. I guess this is due to cross reactivity because in theory my anti-mouse secondary antibody should not recognize the rabbit one, right? Does anyone have a suggestion how to overcome this problem?
Using a secondary-anti mouse antibody that is supposed to detect only non-denaturated antibody (VeriBlot) is not working. I have a very nice band at 55 kDa...
The differences in native proteins and denatures are very small. It's the tertiary and secondary structure which is destroyed. The primary structure is still present. So you can expect, that only a small proportion of epitopes are destroyed during the denaturation. Those small changes can be normally detected only, if you are using a monoclonal antibody.
Second you have to keep in mind, that your protein still have a structure, also after a transfer to the membrane. You are doing blocking ind incubations with physiological buffers, with inert proteins and with detergents ... All three of them leads to some renaturations even on the membrane.
Third: also cross reactions are possible, even after precipitations and other denaturing procedufes ...
So you need antibodies which are really species specific. Use large species differences. (Mouse[rodents] - sheep, horse ...)
have a look here: www.seramun.de
Thank you very much for your suggestions ! Both antibodies were monoclonal but I guess rabbit and mouse might be to similar. I will check if there are other antibodies available
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