Normalization protein for Western blots?

Jose de las Heras via methods%40net.bio.net (by josenet from tiscali.co.uk)
Wed Feb 21 18:43:42 EST 2007


"Peter Frank" <peter_frankde from yahoo.de> wrote in message 
news:d90mt2hnbdb6pddgpu1vd0mpc11hhajtun from 4ax.com...
> OK, I see. This method is not a stripping method in the narrow sense
> of the term  but a method to allow quick re-probing, right? Sounds
> like a good and quick method.

You're right. It doesn't strip the antibody off (it merely inactivates the 
HRP), but if your membrane dries I'm not sure you will get it off at all.
It is quick indeed.

> However, does this also mean that this only works if the proteins to
> be detected need to yield bands that are some distance apart from each
> other, otherwise the antibodies that are still bound would interfere
> with the new primary antibody?

I haven't really tested this, as I don't think I had a case where I wanted 
to see the band in exactly the same position. However I don't think we 
saturate all epitopes when we do a Western, so there may not be an effect at 
all.
For all effects, it's as if you hadn't used the membrane before. You get one 
picture from the first exposure, and another entirely different picture from 
teh second primary.

It's easy to test: have a go, and hopefully you'll like the result.

Jose 




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