The horseradish peroxidase on the secondary antibody produces h202 for the luminol/ECL reaction. Trouble is the H2O2 also destroys the peroxidase. So if you let the blot soak for a long time your killing the enzyme.  Maybe strip , dry , store, reprobe and expose to film.

The instruction manual provided with the ECL reagents will tell you how long the signal persists. The Dura reagents from Pierce claim that the signal is stable for a whole day.

it really depends upon which ECL product you are using.  I use ECL prime from GE healthcare and the signal lasts for hours.  During the first hour, the signal is the same, but it starts to drop off over time.  There were times when the signal was so strong that I left the film cassette in the refrigerator for 4-5 hrs and took a picture and it was fine.  I would call pierce and ask them what the maximum amount of time you have to take pictures.  If you are concerned with timing, you could try using the licor system with flourescent antibodies that will not decay if kept in the dark and the blot has been dried.

Ciao,

I suggest to you to incubate agacondary Ab for 30 min and ahe ECL.  I didn't see any signal after 1h of ECL incubation. So I think you have to expose the nitro to x ray within 30 min or again you can store the nitro in TBST added with NaN3 and then incubate again the secondary ab whenever you want