western blots - survivin - detection of survivin in Panc1 cells (Oct/25/2007 )

Hi,

I was wondering if anyone has done a western blot to detect baseline survivin expression in Panc-1 cells. I have been trying for a really long time without any success.
What i use is a lysis buffer containing Tris , EDTA, NaCl, Brij-97 and Tween 20 for total protein extraction.
use either a Nano-orange assay or a BCA assay to quantitate proteins and then a page gel for the western blot itself following transfer on a nitrocellulose membrane.
Source for the survivin antibody (1:1000 dilution) rabbit so i use an HRP conjugated goat anti-rabbit (1:2000 dilution).

Its really frustrating i see no bands after imaging.
I could really use some help.

Thanks.

Does this Brij+Tween treatment totally disrupt the cell membranes? Or only extracts membrane proteins? To extract survivin you have to open the cell and the nucleus. So I guess that may be one problem. I know nothing about Brij'97, so it may do the job, but Tween is not always aggressive enough to open the cell AFAIK.

Another thing you want to consider is that when you lyse a cell, many proteolitic enzymes are released. Now I dont know whether survivin is susceptible to them (its name suggests that it's a tough guy...), but if you see no bands, you'd better add a few proteinase inhibitors, and make sure they get there in time to stop the proteases.