Why in the world do you need to use super signal, when you get an
intense signal of your sample?
However, I'd expect to see at leat some exposed film at the edge of
the spots. In order to rule out the too strong signal argument, you
might run a blot with serial dilutions of your sample. Just in case.
Did you check the buffers for contamination with azide (will kill
peroxidase activity). If you need to use a preservative, EDTA is the
better choice, though an excess might catch away the iron ions also
necessary for HRP activity.
One way to check for any HRP killing species might be adding aliquots
of any solution that comes in contact with your blot to the reaction
you are using to validate your ECL mix is ok.
> From: Sophie Le Page <slepage at umich.edu>
> Subject: Re: Pierce Supersignal
> Date: Thu, 04 Jan 2001 15:12:29 -0500
> To: methods at hgmp.mrc.ac.uk
>> Recently I have been trying to get the Pierce Supersignal reagent to
> work - with absolutely no success. The films that I develop turn out
> completely blank. The Pierce rep says that this is due to the signal
> being too strong and not lasting long enough for the film to pick up
> the signal.
>> I am using samples and antibody that give a good signal with normal
> ECL. What suggestions do people have as to the dilution factors for
> primary and secondary antibodies? I have tried the whole gamut, from
> 1/1000 to 1/5000 primary combined with secondary dilutions of
> between 1/4000 to 1/50 000. Still with no luck. With normal ECL I
> get a good signal at 30sec with a primary dilution of 1/1000 and a
> secondary dilution of 1/3000. The primary antibody is from Santa
> Cruz and the secondary from Amersham.
>> I am wondering whether somehow the buffer system that I am using is
> interfering with the signal?? For the blocking and washing buffer I
> use 3% BSA in TBST with 0.3% Tween. Antibodies are diluted in 5%
> biorad milk block in TBST.
>> The detection reagents themselves are working because they glow
> brightly when added to straight secondary antibody.
>> Any help would be greatly appreciated as it's driving me up the
This message is encrypted! Use your brain to decode it.
Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de
usual disclaimers apply