I have been attempting to identify a protein antigen of Candida albicans.
The antigen has been partially purified by column chromatography.
Subsequently, the partially purified prep.is separated by SDS-PAGE. The
antigen of interest can then be visualised by Coomassie blue staining. I
would like to generate an antibody to this antigen using mice. A
laboratory manual dealing with antibodies suggests that antigen bands in
acrylamide gels may be used to immunise animals (ie. polymerised
acrylamide acts as an adjuvant). The problem is that the band of interest
cannot be accurately excised without obtaining nearby contaminating
antigens unless the gel is stained. I have been led to believe that
conventional Coomassie staining fixes/denatures(?) the proteins (Acetic
acid is responsible...I think). I would like to identify this band
without altering the protein structure anymore than it already has been
(ie. after SDS denaturation). I have found two methods which may be
suitable. The first protocol utilises 1% Coomassie R250, 1% Acetic Acid
(lower than normal) and 40% Methanol. The second uses a 1% India Ink
preparation made up in water.
I would like to know if anyone can reccommend either of these methods or
reccommend a suitable alternative? Preferably, I would like to stain the
antigen without introducing further variables which might affect antigen
structure or the process of generating an antibody.
Many thanks
Paul
COZZIE
(icostant at info.curtin.edu.au) /| |\
Ph D student / |_| \
School of Biomedical Sciences, / )\ / ||
Curtin University, |---0--- | |-O-|
Perth, Western Australia \ / \ /
6000 ^::::::^
|-O-|
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