Hi everyone! I am having some troubles when developing my western blot membranes using the film system: the films become really dark (even it is difficult to distinguish the bands) when I put them in the developing solution.
It is not a problem of the films because I've already changed them. I was thinking that maybe there is leaking of the ECL solution once I close the box but I've checked this all the time and all was dry.
It is annoying to have those ugly films, and it is very difficult to see the bands.
Thank you in advance!
There is probably a leak of light in your dark room or your cassette . Try changing dark rooms or different cassettes.
Is the whole film dark - or just that area corresponding to your membrane?
Thanks you all for the response. Andreas I'll take care of that, maybe the problem is related with the room as you say.
Hi Rafael, I'll try to provide a photo during the day.
Hi Bruce, it is the whole film that becomes really dark.
OK, then its probably not an interaction between the blocking agent and one of the antibodies. More liklely a light-leak as Andreas suggested
If you put a piece of film directly into the developer and it goes black, then its likely that there is light leakage in the room. Less likely is that the new film is already spoiled.
I've had a similar problem: what I thought was a red safelight was leaking white light at its base. I've also had light leakage under a door! I suggest you turn of all the lights, wait 10 mins in the dark and see where the white light is coming from.
Thanks Geoff! It is the red light that probably causes a mess with my films. I'll put it as far as I can from my films, and in the meantime I'll store the films I am not using in the box, just to avoid any "contact" of the light.
Many thanks to all!
Hi Ana,
You can block the leaking of light from lamp by just putting nontransparent tap (Red or Yellow) at the spot from where it is leaking. Also if you have more than red 1 bulb in the dark room, then you can remove the bulb from faulty, White light leaking socket.
Hope this will help.
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