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Normalization protein for Western blots?

Normalization protein for Western blots? - Protocols and Methods Forum

Normalization protein for Western blots? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 02-19-2007, 08:18 AM
Peter Frank
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Default Normalization protein for Western blots?



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
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  #2  
Old 02-19-2007, 11:45 AM
Jose de las Heras
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Default Normalization protein for Western blots?


"Peter Frank" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

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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  #3  
Old 02-19-2007, 11:11 PM
Anwar Khan
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Default Normalization protein for Western blots?

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
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  #4  
Old 02-20-2007, 12:11 AM
Jose de las Heras
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Default Normalization protein for Western blots?

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

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  #5  
Old 02-20-2007, 02:15 PM
Peter Frank
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Default Normalization protein for Western blots?

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" <[Only registered users see links. ].uk> wrote:

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  #6  
Old 02-21-2007, 11:43 PM
Jose de las Heras
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Default Normalization protein for Western blots?


"Peter Frank" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

You're right. It doesn't strip the antibody off (it merely inactivates the
HRP), but if your membrane dries I'm not sure you will get it off at all.
It is quick indeed.


I haven't really tested this, as I don't think I had a case where I wanted
to see the band in exactly the same position. However I don't think we
saturate all epitopes when we do a Western, so there may not be an effect at
all.
For all effects, it's as if you hadn't used the membrane before. You get one
picture from the first exposure, and another entirely different picture from
teh second primary.

It's easy to test: have a go, and hopefully you'll like the result.

Jose


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  #7  
Old 12-02-2010, 06:48 PM
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Default Re: Normalization protein for Western blots?

I am having trouble normalizing a western blot. I am looking at the expression of a protein in specific regions of the brain, following treatment with drugs. the blots were run as follows...

Control - Drug A - Drug B - Drug A/B

And I have performed quantification on a protein of interest and normalized to GAPDH setting my control value equal to 1.
After replicating the experiment with multiple animals I have data that looks like this.

Control - Drug A - Drug B - Drug A/B
1 - - - - 1.1 - - - - 0.9 - - -- 0.3
1 - - - - 0.9 - - - -0.8 - - - - 0.4
1 - - - - 0.9 - - - - 1.1 - - - - 0.3
1 - - - - 1.2 - - -- 0.9 - - - - 0.5

When I now attempt to run statistics on this I can not run a T-test comparing the control to any of the treatments because I am getting an error saying all the values in one column can not be the same.

What can I do?

Last edited by mcmahoj; 12-02-2010 at 07:39 PM.
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Old 08-04-2011, 03:52 PM
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Default Re: Normalization protein for Western blots?

Do you know if using the Ponceau Red stain technique also works if I'm using florescent secondaries?
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Old 01-04-2012, 12:52 PM
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Default Re: Normalization protein for Western blots?

oh really nice information
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