Re: antibodies for immunofluorescence

Posted by Tamara Howard on
URL: http://confocal-microscopy-list.275.s1.nabble.com/antibodies-for-immunofluorescence-tp7579900p7579908.html

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I'll wade in on this one - I tend to do my westerns on samples that have not been fixed (unless I'm trying to do RIP,  my current bugaboo) and that have been denatured, then run on regular old denaturing SDS-PAGE. I prefer to do immunolocalizations (IF, IEM, immunoenzyme for brightfield) on fixed samples, some of which have also been dehydrated and embedded in paraffin or resin. Some antibodies do not recognize their epitopes once those epitopes are fixed, masked, whatever in the supposedly native environment of a cell/tissue BUT they will bind beautifully to a denatured protein on PVDF.

Now I want to work on Guy's samples - life would be much easier if all antibodies worked all of the time, no matter what horrible/necessary things have been done to the sample!

Tamara
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From: Confocal Microscopy List [[hidden email]] on behalf of Guy Cox [[hidden email]]
Sent: Friday, March 08, 2013 9:02 AM
To: [hidden email]
Subject: Re: antibodies for immunofluorescence

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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 [[hidden email]] on behalf of Martin Wessendorf [[hidden email]]
Sent: 09 March 2013 02:31
To: [hidden email]
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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