Hi,
I am running western blot on serum for IgM and using Goat anti mouse IgM (heavy chain) polyclonal HRP conjugate as secondary Ab (Not using primary Ab, direct secondary). The secondary Ab is against heavy chain i.e. 50 kd product size but i am not sure which protein (75kd) is also picked up by this antibody and its constant. Please check attached image of WB. I have repeated it several times. Kindly share your experience.
Thanks
Anita
Hi!
So you have a heavy chain (50 kDa) and probably complex of heavy chain + light chain (75 kDa)
Hi Timur,
Thanks for suggestion. yeah that could be the reason but the serum sample was denatured with mercaptoethanol that's why i am bit confused.
I read in several publications that some part of ab can be more or less resistant to reducing agents.
The other possibility is that some of the heavy chains denaturated more and because of it moves slower in the gel
Also I am not sure but what if disulfide bonds can be recreated in the gel?
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