chemiluminescence question

Neil Saunders s3003334 at pop3.unsw.edu.au
Mon Jul 31 18:51:50 EST 2000


> I have a question for all of you that use chemiluminescence routinely.
> I am doing transcript profiling by probing dot blots of gene-specific
> amplicons with DIG-labeled cDNA (I'm incorporating the DIG during
> reverse transcription of total bacterial RNA). I'm using an
> alk.phos.conjugate and CSPD as a substrate. So far, signals have been a
> little underwhelming. I have just now tried CDP-star as a substrate and
> signals are better, though someone was mentioning that maybe I should
> think of moving to a peroxidase conjugate and a substrate such as
> Pierce's "North to South".  Has anyone else tried this ? is it better
> than alk.phos./CDP-star ?  I am definitely aiming for the best possible
> sensitivity. Of course if the gains obtained using POD and its chemi
> substrate are marginal, I'll just stick with AP and CDP-star.

Hi Ed,

I think choice of chemiluminescence is one of those personal preference
things, based on individual users experience with their own protocols.  I
started with CSPD a few years ago and after a few "underwhelming" trials, it
came good and I was happy with it.  In my next lab they used it all the time
with modified protocols and had even better results; strong signals after a
few minutes as per manufacturer claims.  I moved lab again and here, they
don't like CSPD much, but are big fans of the peroxidase
conjugate/substrate.  Sure enough, my CSPD is underwhelming again, but
improving all the time!  I can't think of a good reason why POD should be
intrinsically better than AP, I think it's just a case of optimising your
particular blotting and detection protocol.

Neil
--
School of Microbiology & Immunology,
University of New South Wales,
Sydney 2052,
Australia

Ph: +61 2 9385 2093
Fx: +61 2 9385 1591
email: neil.saunders at unsw.edu.au
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