SCBT Tech Support

Okay, I found the problem.  It was simply that the gels I was making weren't working with my samples.  Pre-cast gels work fine, and I get nice, clear, bright bands where Bcl-2 is supposed to be.

Odd thing is, my ladder worked even with the gels I was making.  And using fresh SDS didn't help (ALL my reagents for casting gels were purchased within the last 2 months, except for the tris).  I'm really not sure why my gels weren't working, since I've made hundreds of gels before in other labs and they've always worked fine.  But I also don't really give a damn, I found something that works.  

Now I've got a contamination issue in my organ extracts... but hell, that's a way better problem to have than bands not showing up when they should, so I'm still happy.

Thanks for the help everyone!

The beta-actin mouse monoclonal AC-15 from santa cruz is very good.

I think there is a little confusion regarding the HRP versus the ECL.  The HRP (horseradish peroxidase) is an enzyme that oxidizes a substrate.  This oxidation reaction with chemiluminescent substrates produces light.  The HRP enzyme can remain active as long as it is properly stored as previously mentioned (refrigerate in buffer, no azide).  Every once in awhile I'll have the enzyme die and then all you need to do is reprobe with the secondary antibody but usually the HRP remains active for days even after extended exposure to ECL.  ECL contains the HRP substrate as well as "enhancing" chemicals, usually modified phenols.  So, the moment you expose your blot with the HRP-conjugated antibody to the ECL, the substrate begins to be used up but it doesn't destroy the HRP enzyme.  If you need to store your blots for awhile (more than a few days), be sure you change the buffer.  If bacteria begins to grow in the buffer, the pH will drop and destroy the HRP.  But if it does, or if the signal is too weak, just reprobe with the secondary antibody or protein A-HRP.