Normalization protein for Western blots?

Anwar Khan via methods%40net.bio.net (by akhan357 from sbcglobal.net)
Mon Feb 19 18:11:46 EST 2007


Jose, did you mean to say that instead of stripping the blot (by some other 
method), just add poncaou, wash, block and use the blot second time for 
another antibody?  I assume the ponceau buffer kills the enzyme on secondary 
antibody? if this works well, thank you for sharing this.
regards,
Anwar
"Jose de las Heras" <josenet from tiscali.co.uk> wrote in message 
news:53th5sF1smqdbU1 from mid.individual.net...
>
> "Peter Frank" <peter_frankde from yahoo.de> wrote in message 
> news:fgmit257taai3nfooolud92l4fj5ggb4h4 from 4ax.com...
>> Hi,
>>
>> when performing semi-quantitative analysis of Western blots, it is
>> supposed to be a good idea to use a normalization protein, i.e. a
>> protein that is generally not regulated. I heard beta-actin is not a
>> good choice.
>>
>> a) Can you recommend a good normalization protein for use with
>> semi-quantitative analysis of Western blots? (This also means that
>> appropriate antibodies should be commercially available.)
>>
>> b) In order to perform the normalization, the Western blots will have
>> to be stripped and reprobed with the antibody for the normalization
>> protein. Can you recommend a good stripping procedure for Western
>> blots? I tried stripping in 0.2 M NaOH for 30 min at room temperature
>> and it didn't work very well. I did not let the blots dry out. But the
>> overall background was rather high after the reprobing and the
>> reprobing efficiency was lousy compared to a first-time probing.
>>
>> Regards,
>> Peter
>
> Hi Peter,
>
> if you're concerned about the suitability of one particular protein as a 
> control, you can always use more than one, and test how well they perform. 
> b-actin is commonly used as a loading control, also some form of tubulin 
> (in general cytoskeleton proteins are used for these purposes). We haven't 
> done extensive tests, but are generally happy with actin. I have been 
> doing microarrays for a while, and I have checked how b and g actin 
> behave, as well as several tubulins and other proteins like GAPD that ar 
> used commonly as controls in RT-PCR, and they tend to behave quite well 
> across experiments, meaning that if there is an efect, it is small, or it 
> affected the whole set of experiments in a similar manner. I think it's 
> good to question these things, and test whether your "control" protein 
> actually acts as a good control, but I think in most cases they will 
> behave ok.
>
> As for stripping, we simply pour some Ponceau solution (the red stuff used 
> to stain the proteins on a western blot and crudely check the transfer 
> went ok, loading, etc) and after about a minute we remove it, then wash 
> the blot with distilled water, and block again and apply your new primary. 
> It is a very simple procedure and works very very well. You can strip 
> several times, I've heard. I only ever stripped twice, and usually only 
> once. It's always good. Just make sure you wash the Ponceau off.
>
> Jose
> -- 
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