Primary Mouse antibody on mouse tissue
Posted 09 February 2011 - 01:20 PM
I have mouse tissue as starting material to extract protein and run western.
The problem is, the antibody I use to detect the target protein is also from mouse.
That means, when I use a 2nd antibody, it has to be rabbit anti-mouse, goat anti-mouse, etc. And such 2nd antibody will always give me a signal, near 70-80kDa (this signal will be there no matter what, even if I do not add primary antibody)
This signal is in fact, I believe, endogenous IgG from the mouse cells.
So how can I solve this problem?
The quick fix is of course switch to a non-mouse primary antibody, but what if that protein I want to study only has mouse primary antibody??
I really need some advice.
Thanks a lot!!
Posted 11 February 2011 - 12:17 PM
if so, does the target protein migrate far enough away from the endogenous protein?
if so then you can ignore it (or use it as an internal standard, if it is consistent).
if no for either question then you should find a primary from a different source (if no for the second question then you may be able to deplete the interfering protein before applying to the gel but that is usually inconsistent).
Edited by mdfenko, 11 February 2011 - 12:18 PM.
genius does what it must
i do what i get paid to do
Posted 15 February 2011 - 11:36 AM
If that suggestion really isn't working out for you another option you could try (like mdfenko suggested) would be to deplete the interfering protein. Depending on the sample size that you want to process there are some efficient and reproducible techniques you can try to deplete that IgG from your sample. I have found that samples under ~10 uL work best.
Edited by proteaMatt, 15 February 2011 - 11:40 AM.