Normalization protein for Western blots?
Jose de las Heras
(by josenet from tiscali.co.uk)
Mon Feb 19 19:11:20 EST 2007
Yes, Anwar, exactly that: just add Ponceau for a minute, wash it off, block
and use again.
In our hands (it's the method we use in my lab, not just me) it works very
"Anwar Khan" <akhan357 from sbcglobal.net> wrote in message
news:S8qCh.15724$O02.4161 from newssvr11.news.prodigy.net...
> 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.
> "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...
>>> 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.
>> 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
>> Musha ring dum a doo dum a dah - www.mcnach.com
>> Current fave guitar: Fender 'Sambora' Stratocaster
>> Fender Stratocaster - part coffee table, part spaceship.
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