Western Blot with ECL

Alex chen.1069 at osu.edu
Mon Apr 26 17:31:17 EST 2004

Dear all,

I now do Western blot in mouse system with ECL method. I got very high
backgrounds when using 1st antibody, followed by 2nd antibody. So I
did the troubleshooting. I applied the 2nd antibody only to the
pre-blocked strips and detected. The second antibody is conjuncated
with AP. And I get substrate from Molecular Probe. I get a very high
background at the concentration below 1:30,000. But get less or none
in further dilution up to 1:300,000 and 1:3,000,000. I have not tested
for the first antibody yet. I wonder if someone out there uses such
diluted 2nd antibody?


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