When I am trying to develop my blot by ECL method, I am facing this problem. After all the antibody incubation and washes, when I am adding luminol/H2O2 on to the blot, I could literally see the glow. But after keeping it in the casset ( In the usual method, I put the blot on a plastic sheet, then put an another transparent plastic sheet on top of it and then keep the film) and after exposing for different times (10 secs to 5 minutes), I could only get a blank X-ray film after developing and fixing it. The same blot, when I developed with TMB reagent, came off very well. My antibodies are working well because otherwise I woudn't see the glow or wouldn't get it with TMB/H2O2 reagent.
I tried to troubleshoot in many different ways, changing the X-ray film, changing developper and fixer, varying the exposure time, changing the antibody dilution, preparing all the reagents fresh, but nothing worked so far. Earlier I used a commercial ECL reagent from Thermo Scientific and then switched to luminol and coumaric acid prepared in the lab. This reagent is used by my other labmates and works well. I prepare H2O2 just before use.
I got upset and now developing with TMB only, since I am running out of time.
Any points/suggestions which I might have missed will be highly appreciated.
Looking for a solid reason/suggestion, not just a vague, generalised answer.
Thank you!
Yes.. if tmb is working and yu have ample amount of abs, just get through with it.. don't waste yur time and energy in researching ecl protocols unless yu lay yur hands on chemdoc..:P
Thank you, Dr. Hoda. Being an earlier labmate, you know exactly what needs to be done. :)
If you are able to see the glow in the membrane, there is no way the bands could not appear in the x-ray sheet. Here, the problem is solely with the x-ray sheet that you are using or the developing solution. Cut a piece of x-ray you are using, expose that to light and develop it and try to find the cause of the trouble.
It's better to go for chemidoc while you are using ecl like Dr. Hoda mentioned.
It seems that ECL is working perfectly but issue with developer and fixer. You have to develop x ray film first and then fix it. If the problem persist, you must try developer and fixer of the different company.
Thank you Dr. Sankar for your concern and suggestion. I also did what you said, anna. I Took a normal film, developed it, it appeared transparent, as was my membrane-exposed film. Then, I took a film, exposed it to white light and then developed, it appeared as completely black. Then I covered a small portion of the film and then exposed the film to white light. The portion of the film where I covered during exposure to white light, appeared transparent and the rest of the film appeared black after developing. That means the X-ray film and the developer-fixer is working properly. I don't really know what am I missing here.
Good that you analyzed all those stuffs. You should surely get the bands. I couldn't understand what could be the problem. However try to buy some new x-ray sheets. I will let know which one to buy. If you are getting results with tmb/h2o2 then better continue with that for time being.
I had that problem once, and it was the x-ray film's fault. They seemed to be ok but weren't, I was also getting dark films when exposed to light and everything, but did not work with my bands. After buying new ones the problem disapeared, still not sure of what was wrong with the films, but obviously they had some kind of flaw.
Thank you, Dr. Samanta. I am going to get new X-ray Film and see what happens.
Thanks everyone for all of your concerns and suggestions/sharing your thoughts. I bought new X-ray films and its just working fine. Now I am sure the problem was with x-ray films only. Thank you Dr. Samanta. Also, came to know that the blue-sensitive X-ray films works best with ECL. The one which I bought now is blue sensitive fast laser X-ray film from VMS company.
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