On Sep 23, 4:22 pm, WS <[email protected]> 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 -- Nick Theodorakis [email protected] contact form: http://theodorakis.net/contact.html _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
