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ECL visualization method in WB


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#1 Lee6948

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Posted 26 December 2010 - 07:40 PM

Hi,

I used to use CCD method to visualize my western blot results, which was fast and money-saving and allowed me to choose exposure time amd strength. But now I'm working in a hospital where everones uses the ECL method. I often have problems of overexposure or photos washed inadequetly.

So, does someone know how to do this ECL thing correctly?

#2 BioMiha

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Posted 27 December 2010 - 04:08 AM

Hi,

I don't know if I got this right... You were using a CCD camera (CCD being a type of sensor chip used in cameras)? Now you are developing WB on film?
ECL stands for enhanced chemiluminescence, which you can detect either on camera or on film. Based on the experience I have, I'd say it depends more on the amount of protein you loaded and how you ran your gel than on the film development. However, as I suppose you've done many WBs before, so I won't go into that. For film development I usually use a fairly sensitive substrate (SuperSignal West Pico from Pierce), unless I loaded a really small amount of protein in which case I go for the heavy duty stuff (ECL Ultra from Perkin Elmer). I am going to assume that you have a dark room and a cassette for film development. I usually expose the film for 2-5 minutes and see if it works. If it's too saturated I expose for less, otherwise more. I really think fresh developer is important. We used to reuse it over and over and over again and I saw the same in other labs. But when it becomes yellow or brown it really stops doing its thing so it's best to replace it with a fresh batch. I have also started using a piece of scotch tape to tape down my film over the membrane, so it doesn't move when I close and open the cassette. If you have a really sensitive film even a couple of seconds of wobbling will result in bands all over the place.
So in steps:
Apply the substrate.
Drain the membrane (do not dry).
Put the membrane a plastic wrap, put it in the cassette and take into the dark room.
Put a piece of film over (tape optional).
Close the cassette.
Time 2-5 minutes (less if needed, more if needed) with a stop watch.
Reopen the cassette. Take the film and throw it into the tub of developer solution.
Shake evenly so the developer can do its thing.
Using forceps, take the piece of film and throw it into a tub of plain water.
Shake a bit to remove developer.
Place the film into a tub with fixer solution.
Shake to fix the film (you will see it changes colour).
Put the finished film into the tub with water again.
Shake a while to remove the chemicals of the stripped film.
Dry the film. Do not scan or place the film anywhere untill it's dry because it is really sticky.
Enjoy the fruits of your labor.




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