Primary Mouse antibody on mouse tissue - How to use mouse antibody to do western on mouse protein (Feb/09/2011 )

Hi,

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!!

does it detect the target protein?

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).

When you prep your sample for the gel do you denature it? I run a lot of gels with sera and when I am specifically looking for IgG, I skip denaturing steps (BME or heat) so that the IgG will not break down into the light and heavy chains. This will allow it to stay up in the 160 kDa range on your gel. If you let your gel resolve enough perhaps you can physically separate your target protein far enough away from the IgG. If you do skip the denaturing step you might need to adjust your percentage of acrylamide. I have found for sera that I run a 7.5% acrylamide gel seems to be a happy medium, it allows you to stack your gel and still allow your denatured proteins to enter into the gel.

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.

Good Luck!