Hi there,

You might need to strip and reprobe the membrane to get the signal.

western blots should be developed within an hour of applying ECL, because the signal does indeed fade away over a relatively short period of time; it doesn't matter how you store it. you might add some more ECL, but as Dominique said, your results will be better if you strip and reprobe

It is normal and sometimes happens that you can see a chemiluminescnece emission but I do not know why you had problems with recording it. I guess, the next day you used ECL once again because you washed it away with TTBS. However, as the others wrote, it's better for you to strip and reprobe your membrane ;)

:-) ECL light emission is only transient...the home made (luminol and H2O2) vanish pretty fast and some of the various suppliers last .;say longer..but as others recommend..if you expect a strong signal , dip the membrane in the lab and take your time to go to the imager or dark room...on the contrary, if you expect a faint signal ..develop your NC/PVDF in the dark your or next to the imager...and do not forget...ECL light rapidly saturate film silver grain...so better use a CCD camera or go for odyssey (IRDye imaging)..

Thank you all very much for taking your time🙏. I agree there is some ambiguty in my question.

In the first time I had a new unstandard (probably) developer buffer which made the film completly black within 1-3 seconds. So I couln't record my signal and decided to record it at the next day, I rinsed the membrane to remove the ECL before putting the membrane in -20. The next day again I used fresh ECL substrate twice from 2 different manufacturers.

What I saw in the dark room seem to be a strong signal, anyway I have no other option! recording ECL on film is a luxury for me 😊.

I am wonder whether the HRP enzymatice activity can be destroyed after storage?!

How I can strip my membrane? Is there any protocol you can recommend?

Here u have two protocols from Abcam. I use the first one.