Western for membrane bound protein - (Nov/27/2007 )

Not sure if this will help... but I am also working on a membrane bound protein which I extract from tissues... I use a simple Tris-EDTA (TE) buffer plus protease inhibitors when homogenizing the tissue. After that I spin down at low speed (1000cfg) to remove cell debris and then collect supernatant and re-spin at 12000cfg to obtain a crude membrane fraction. I cannot get a signal for the membrane protein in a crude total protein lysate. You probably can store your protein lysates in TE buffer and only add reducing buffer into the sample that you need to run a gel with...
Sindy

The Best way to do it without harming your protein is this.

3. Add 1% DDM to the lysate.
4. Run solubilized lysate on gel.