Normalization protein for Western blots?

Peter Frank via methods%40net.bio.net (by peter_frankde from yahoo.de)
Tue Feb 20 09:15:40 EST 2007


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.

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?

Peter


"Jose de las Heras" <josenet from tiscali.co.uk> wrote:

>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 
>well.
>
>Jose
>
>"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.
>> 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
>>> -- 
>>> 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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