Preparing mouse brain lysates for western blotting - (Aug/16/2008 )

Hi,

I have been trying to detect the expression of ZIC3 (transcription factor - nuclear protein) in adult mouse brains.

The only method that I can really use is SDS-PAGE + Western Blotting. However, I have been having immense difficulties in detecting the desired protein through Western Blotting. The antibodies that I use are not an issue as I have tried them on in vitro systems, where they work perfectly fine. When I use these antibodies on my brain lysates they come up with a lot of unspecified bands, rather than my desired band. I suspect that my method of lysing the brain tissue might have something to do with it. Currently, I am using a simple lysis buffer (contains 1% NP-40, Tris, MgCl2, etc.). I feel that this buffer is unable to extract enough protein from the nucleus, and therefore the antibody cannot recognize its presence.

Please inform me if somebody has any ideas/suggestions on a different lysing method, or even any other solution.

Thank you in anticiaption,
Jerry

In vitro is different than in vivo where you can have diverse modifications of you protein.. most likely your extraction works fine. I would be worried more with issues like.. from where are you extracting your protein? hippocampus? cortex? your protein might have different expression patterns and modifications in different regions.
do you have a control as in a knockout mouse tissue? would you think of another control you can use?
so many questions so many dilemmas

I agree with tertu, but i agree also to the fact that your buffer may not be adequate for nucleus extration.

Try a RIPA buffer, with Triton, sodium deoxycholate and BME. The recipe should be found pretty easily.

Good luck!