western blot from heart lysates - membrane extractions too (Nov/05/2008 )

Hi everyone
I was wondering if anyone has experience of doing westerns from whole heart homogenates. I've been homogenising in tris with triton, PMSF and protease inhibitor tablet and have been loading around 25ug protein but my bands for beta-actin are very weak. I'm starting to wonder if my homogenisation is effective.
Also, does anyone do membrane extractions from mouse heart? I have been told you need a lot of tissue for this but has anyone done this successfully from a single heart?

I would be grateful for any suggestions!
Thanks
Alison

actin is usually locked up in filaments. if you just homogenize and spin then you will pellet most of the actin.

you can either depolymerize the actin (with atp) or prepare your homogenate under denaturing conditions (sds & 2-me or dtt).