Normalization protein for Western blots?

[Jose de las Heras]

"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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