Normalization protein for Western blots?

Jose de las Heras via methods%40net.bio.net (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 
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.
>>
>>
>
> 




More information about the Methods mailing list