Hi Guy,

I don't think what Martin said is at all controversial.  Some antibodies are very good at recognizing epitopes that are unmasked only after proteins have been denatured by SDS-PAGE, making them useful for western blotting.  Other antibodies can recognize epitopes that are present in the native protein, making them useful for IF, but epitopes can be destroyed by denaturing, which makes antibodies that specifically recognize them bad for westerns.  As long as you have appropriate controls to show the specificity for each antibody in each assay, I see no reason why one couldn't use two different antibodies for the same protein, depending upon the application.

Eric


Eric Griffis
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On 8 Mar 2013, at 16:02, Guy Cox wrote:

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Martin,

             I must say I am very worried by your statement "Antibodies that work for one application (e.g., westerns) may be completely unusable for another (e.g., IF)".  Can you give me a biological reason for this?  It would seem to make most cell biological work impossible.  I would assume that most of us work the same way as me - ie using microscopy to identify the location, and chromatography to characterise the protein.  If you don't use the same antibody both times you will just be generating nonsense!

                                                                             Guy

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From: Confocal Microscopy List [[log in to unmask]] on behalf of Martin Wessendorf [[log in to unmask]]
Sent: 09 March 2013 02:31
To: [log in to unmask]
Subject: Re: antibodies for immunofluorescence

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Dear Michal--

On 3/8/2013 2:36 AM, Michał Majkowski wrote:

I do not know if this is a good place to address my problem as it
regards immunofluorescence. The problem is: we have in the lab
antibodies dedicated to Western Blot (according to manufacturer; Abcam).
We used them to IF and we obtained some nice images. What do you think
about such data? Should it be confirmed by IgG dedicated to IF? Thank
you for help.

The quick and safe answer is that you can never trust any antibody for
any purpose without first characterizing it thoroughly.

Antibodies that work for one application (e.g., westerns) may be
completely unusable for another (e.g., IF)...or they may work perfectly.
 A starting point is to compare the labeling that you obtain, to the
labeling that's previously been published.  The "gold standard" is to
demonstrate that the labeling is not observed in a knockout animal.

Cliff Saper at Journal of Comparative Neurology published an editorial
on antibody use that may be helpful.  It can be found at:
http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .

Good luck!

Martin Wessendorf

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