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
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
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 wall!!