Alkaline Phosphatase Probes

Nick Theodorakis via methods%40net.bio.net (by nick.theodorakis from gmail.com)
Thu Sep 23 20:42:55 EST 2010


On Sep 23, 4:22 pm, WS <novalidaddr... from nurfuerspam.de> wrote:
> 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.

What might be easier is to get a biotin incorporated into one or more
the nucleotides in the oligo. It's a commercially available option in
custom synthesis these days so the OP won't have to do the chemistry.
Then detect with streptavidin-alkaline phosphatase conjugate (also
commercially available).

Nick

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