Different detection methods (ECL and AEC) on same blot - (May/15/2008 )

Hello,

Is it possible to first detect proteins on a blot with ECL and afterwards with a colorimetric method?
Do I have to strip the blot and with what buffer?

Many Thx

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I have never done it, but I am pretty sure you can do it in a succssive steps.

Do one, strip the blot, do the other. I don't know which sequence would be better.

For Stripping: http://search.vadlo.com/b/q?keys=stripping...mp;sn=158621799

..

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do the ecl first, then the colorimetric. the deposited color may quench the ecl if you do it the other way (and the color doesn't strip well).

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OK Thx. But do I have to strip and start all over again by blocking and antigen incubations or can I just wash after ECL and try AEC?

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in general, if the enzyme conjugated to your secondary antibodies is different then you don't have to strip.

if they are both hrp then you can kill the enzyme from the first secondary with sodium azide or with peroxide, wash thoroughly and go on.

if you are looking for the same protein then, to be safe, you should strip and start with blocking. (because you don't know if the hrp from the first secondary survived and the antibody will be occupying the binding sites that your new secondary will try to find).

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