Delution of detection reagent? - (Feb/07/2005 )
I'm using the 'Chemiluminescent Peroxidase Substrate for Western Blotting' by Sigma for detection of my protein bands. Only problem is that this substrate gives *such* a strong signal. Has anyone ever 'diluted' this substrate and if so ... in what can I dilute it to get less strong signals?
I'd try to lower the used antibody concentrations, not to dillute the ECL-Substrate...
Otherwise you can try to add the reagent and put the film on the membrane sometimes later. Most ECL-reagents will produce a stable signal over 5-10 minutes that then decreases....
You can dilute ECL reagents in Deionized water. Try diferents dilutions but mantain the correct relation between the reagents.