Alkaline Phosphatase Probes

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Thu Sep 23 16:22:34 EST 2010


Hi Yvonne,

some possibly very weird ideas (sorry, I'm a chemist):

a) any chance to have a carboxylic group attached to the oligo? Then
activate the carboxylic group by a carbodiimide method and couple to
lysine residues of AP
b) any chance to have one ribobase incorporated into your oligo? Then
do periodate oxidation and link to lysines of AP, as described in
standard protocols for HRP-labeling (it's just inverse, this time)
c) any chance to get streptavidin attached to AP? then buy
biotinylated oligos, mix and you're done. In worst case, just try to
couple streptavidin and AP with a bifunctional cross-linker.
d) as thiol modified oligos seem to be available (http://
www.metabion.com/products/dna_modifications.php), use a maleimide/
succinimide bifunctional linker to attach it to AP: Maleimide
activated carboxyls react with SH groups, succinimide activated
carboxyls with aliphatic amino groups. Thus, first react the oligo
with the maleimide part. If desired, purify by gel filtration (either
absolutely water-free or in 1mM HCl), then react with AP in 100mM
Borate or Carbonate, pH8-9. Tris or similar aminogroup compounds as
well as BME, DTT etc are absolutely to avoid, of course, they'll screw
up the couplings.

Hopefully, these thoughts will lead you to something feasible...

/Wo

On Sep 23, 4:29 pm, "Yvonne Couch" <yvonne.co... from pharm.ox.ac.uk>
wrote:
> Hi all,
>
> I'm in the UK and am looking for a company that might sell alkaline
> phosphatase labelled oligonucleotides for in situ hybridization - anyone
> have any ideas?
>
> Thanks
>
> Yvonne



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