Cloudy Western - (Aug/21/2008 )
Hello! Can you help me with this western blot problem?
A big part of my films ends up being cloudy when I develop them; like in this picture:
(attached in next post)
And every once in a while, under the exact same conditions, some of them will be decent:
attached in next post
I thought I had previously optimised antibody concentration, incubation times, etc. I do the following:
- PVDF membrane prepared according to the manufacturers info(including the metanol step)
- 3h block in 5% dry nonfat milk in PBS-T
- 1h incubation 1:5000 primary
- 1h incubation 1:20000 secondary
- 5 min with Pierce's Femto detection reagents (I use a diluted mix (75uL each reag to 2mL water) )
- 5-10 min exposure time
And when the film does not become cloudy, I get a nice signal. It seems like the cloudiness is independent of signal, since I normalised the protein in my gels and I used the same batches of reagents and developing solutions (none of them after expiration date) for both images (couple of days difference between blots!). Sometimes it happens, sometimes it does not.
So I guess it must be something really really basic that I'm doing wrong, or not doing, unconsciously, in some blots. Has this happened to anyone else?
PS - I'm paranoid about not leaving any air bubble when I transfer (semidry system) or when I incubate with blobking buffer/antibody. I handle the film and membrane with gloves and there's no light reaching the films.
Your pictures did not post correctly but it sounds like it's your developer. If your film takes on a cloudy/white almost powdery cast, your water in your developer is not circulating or being replaced often enough. What you are seeing is the fix that did not wash off and this will destroy your image. Does it get worse with time? Usually when there is a problem the film looks only slightly cloudy at first but quickly gets worse and worse. It is important that the film developer is taken apart and cleaned from time to time.
Thank you for your reply.
I attached the pictures in this post so you can see them.
What do you mean by the water circulating in my developer?
What I do is this: After incubation with detection reagents, I slip the membrane in a transparent plastic bag and expose it to the film for the time I want to (in the dark, inside a black box) and then I quickly submerge it in the developer solution; as soon as I see the bands I quickly tranfer it to a fixing solution, and after a bit to water to clean it off.
We buy these solutions and because we reuse them (we're poor!), after a lot of blots the developer will indeed not be so clear. Usually when I dip the membrane in the developer I can already see that it will turn out bad. It does get worse with time in the developer, but not after fixing the film... is that what you mean?
It looks like a problem with the chemicals being used to develop the films. You can see the chemicals streaked across the film. Are other people having any problems with their films? Sorry but I'm not too familiar with developing film by hand. We have a machine that pumps in all the chemicals and water. You just feed in the film but I'll start to have problems when the chemicals are old, running very low or there is a problem with the water supply to the machine.
I've had this problem occasionally too. I can't say for certain, but I think that it has to do with the Femto kit. It's really sensitive, and if you don't get all the solution off the membrane then it has a greater tendency to do this. Obviously you can't let it dry completely, but I try to get as much off as possible. I usually let the solution run off the membrane onto a piece of paper towel, and also shake the blot a few times before wrapping it in saran before I set it down on the film. If you get the cloudy picture, you can try to wash your blot a few times in PBS, then redo the ECL steps.