Sensitive alkaline phosphatase assay?

Bernard Murray bpmurray*STUFFER* at socrates.ucsf.edu
Mon Jun 22 04:46:32 EST 1998


In article <358BC047.6FB2 at worf.molbiol.ox.ac.uk>, South, Parks, Road,
Oxford, OX1, 3QU, United, Kingdom. wrote:

> Does anyone know of an assay for alkaline phosphatase that is more
> sensitive than the colorimetric (i.e. p-nitrophenyl phosphate) one? A
> radiochemical one, perhaps? or could I adapt one of the chemiluminescent 
> southern/northern protocols? A problem I see here is that the
> luminescent products are (necessarily) insoluble wheras for an enzyme
> assay it would be better if it were soluble... 

[snip]

> Dudley Page (mpage at worf.molbiol.ox.ac.uk)

Although the chemiluminescent substrates are used in blotting etc.
protocols they can still be used in liquid assays.  I've dabbled
with Clontech's SeAP (secreted alkaline phosphatase) assay which
employs CSPD as the substrate and you should also be able to use
the more recent versions (eg. CDP-Star).  I used a standard
tube luminometer method (and didn't have any success with the
"put the solution in a microtitre plate and expose to film"
method).
    I believe there are substrates capable of generating
fluorescent products and if you have a FluorImager/Storm
you may be able to adapt your assay to a dot blot or drop blot
format and use their chemifluorescent substrate(s) (eg. Attophos).
     Good luck,
          Bernard
-- 
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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