Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Thu Jun 2 06:53:05 EST 2011
In article <mailman.977.1304214922.20886.methods from net.bio.net>,
7lightbourn from gmail.com says...
> I would like to know what technologies can be used to identify visualize or
> quantify new alternatively-spliced isoforms for which antibodies are not
> available. Alternatively, I am curious as to how such antibodies could be
> made cheaply in the lab.
Since you say the gene is cloned, you know how the splice-isoforms
differ in amino acid sequence. It would therefore be a simple matter to
synthesize short peptides that are unique in those isoforms. Those are
then linked to a carrier protein (e.g.., keyhole limpet hemocyanine) and
injected into animals to generate mono-specific (but polyclonal)
In my experience, chicken are ideal for this, maintenance is simple and
cheap, and the antibodies are isolated in large quantities (about 100 mg
each) from the egg yolk (called IgY, but really it's just IgG). Only
disadvantage: IgY do not react with protein A or G, you need a
(commercially available) anti-IgY antibody instead. Isolation of IgY is
very simple, the yolk is diluted 10-fold with PBS and a 35-120 mg/ml
PEG-6000 cut is prepared. The crude preparation is dissolved in PBS
again and then further purified by a 25-50% ammonium sulphate cut. The
precipitate stores well suspended in 50% AS in the fridge.
For the peptide synthesis it is more economical to have it done
commercially (or in a core facility) than to mess around with it
oneself. Just one thing to remember: Merrifield peptide synthesis
proceeds from the C-terminal end, thus peptide chemists give sequences
in wrong direction (C to N).
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