Western Blot with ECL

Jayakumar, R R.Jayakumar at RoswellPark.org
Tue Apr 27 08:55:58 EST 2004

Generally the idea is the you dilute your antibodies 5- 10 fold more than normal dilutions used with sensitive substrates like CDP-star or the ones used in ECL (ie., if you were using 1:10000 dilution, you must use 1:50,000 to 1:100000 for very sensitive substrates like CDP-star etc.).  this is when compared to the relatively weaker substrates like CSPD. I am not sure what sort of substrate you were using earlier, nor the antibody concentrations you were using before.   So if you were getting high background with ECL, then the most likely suspects are the antibodies, unless otherwise you are doing low blocking and low stringency washings.
    bye and best of luck


> ----------
> From: 	owner-methods at hgmp.mrc.ac.uk on behalf of Alex
> Sent: 	April 26, 2004 6:31 PM
> To: 	methods at hgmp.mrc.ac.uk
> Subject: 	Western Blot with ECL
> 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?
> Alex

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