If you could see the bands on membrane but you cant see in xray films then check the x-ray filmes, if you see burst of signals and the signal disappears then use low concentration of secondary AB.

Did you get any halo/ghost bands (white regions) in your film? Then probably, your protein of interest would be very abundant and you may want to consider loading less amount of your sample; OR you might be loading too much. These halo bands does appear when using sensitive chemiluminiscent detection systems.

You can also try less conc. of secondary Ab as Ali has suggested.

Good luck

You said : "signal was seen on the membrane". Do you mean, you got to see brownish colouration of your bands on your membrane after trying to develop the film? Then probably its a case of overloading your protein. Also you can try less exposure of your film. what exposure have you been doing ?

if you think you are getting ghost bands, then this paper would be some help to make your understanding better.

http://www.piercenet.com/files/AN0011.pdf

i was using the x-ray film earlier. it was working well. moreover its a new one i purchased.

I have seen a flourescent light on the membrane but do not get bands on x-ray. There were no ghost bands seen. The film was clear.

Hi Pallavi, what do you mean by you could see a band on the membrane? Do you mean such a brown staff? If this is the case, your signal is to strong leading to burning the membrane itself because usually you shouldn't see your band on the membrane by chemiluminescence just by staining it with Ponceau for example before blocking. If your signal is such strong, that you burn the membrane you will see the band as a white spot on your film in case you have high background. Yes, as a WHITE band (not black as usually). Leading to the observation that you do not see anything if your background is very weak.

no, actually i didnot see band on membrane. I mean i see the flourescent light on membrane after putting ECL reagent. then it was not developed on film. Since on membrane i get the flourescent light, that means upto that step, everything is allright, then what makes that the bands not seen on membrane. I have taken the fresh developer. It was clear light yellow color.

If anybody had faced the similar problem, and was able to solve it, please guide me too.

I think that is your antibody's problem. All protein can loading on the gel and transfer on the membrane. what kind of primary antibody with second antibody decide your result. You can got band by advance ECL. It means your antibody was not enough or sample protein's concentration was not enough.

how can i check if my primary and secondry antibodies are working well?

When I used ECL to detect, I dilute primary antibody 1:1000; and second antibody 1:10,000.

Advance ECL: I dilute primary antibody 1:5,000; and second antibody 1:100,000. I always apply sample (protein concentration)15~20 ug/line.

So I think you need use several type, such as from 1:500, 1:1000.... And find which is good.