Re: antibodies for immunofluorescence

Posted by Martin Wessendorf-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/antibodies-for-immunofluorescence-tp7579900p7579910.html

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Dear Guy et al--

On 3/8/2013 10:02 AM, Guy Cox wrote:
>                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!

The environment in which a protein exists in a western is quite
different from that in which it exists in the cell: in a gel, it hasn't
been fixed (and thus chemically modified) and it may have been subjected
to detergent treatments and other steps that denature it in ways
different from those used in fixed cells or tissue.  For this reason,
the best way to characterize an antibody is to do so using the same
methods in which it is to be used--anything else requires making
assumptions that may or may not be appropriate or accurate.

In the experience of my lab--and I expect in the experience of the
antibody vendors as well, given the fact that they carefully label
antibodies as being useful for one purpose and not another--there
sometimes are antibodies that work for westerns that don't work for
immunofluorescence, and vice-versa.  We also find antibodies that appear
to label a substance specifically in cells or tissue, but that label a
variety of bands in westerns, some of which don't appear to have a clear
relationship to the substance in question.  I think this gets to be more
of a problem when dealing with proteins that aren't very abundant and
when the antibodies are raised against epitopes that are common to a lot
of other proteins.

In any case, my point is not that antibodies that work well for westerns
CAN'T work for IF.  It's that they may not.

Martin
--
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