Hello,
Today i want to ask about films used for detection during western blot.
When i conduct detection, i know quite well that film should not
be in developer solution too long to avoid burning and i have to make sure all parts of film should be soaked with developer and fixer.
I often put multiple films on developer or fixer at the same time(ex)when signal is too strong,
I put two films in stack above ECL-treated membrane)
and i wash them with water and dry them.Then i use them as final research outcomes. During this whole process
i want to know whether there are situations when detection result of specific film is affected by another film detected.
Thank you very much!
1) be careful when placing multiple pieces of film into the developer, water rinse, or fixer as if they stick together, the image may not develop properly.
2) I am not sure placing multiple sheets of film on the membrane for the same exposure is the best way to go. It may be better to do multiple exposures of various times. You can tell within the first minute in the developer if you need to do a longer or shorter exposure or even switch to a film that is more or less sensitive. One of the benefits of developing it trays, rather than an automated developer, is you can optimize your exposure times more rapidly.
Good advice from Bruce. Another tip that I often use when the membrane is too 'bright' is taking Kim wipes and pressing down evenly on the membrane to remove excess reagent (of course without letting it dry). This usually gets the signal down and removes background. Maybe you can circumvent the need to use so many films. Otherwise, back off on antibodies systematically.
All good advices. Sometimes it can also help to quickly rinse the membrane with water, to further remove excess reagent, if such remains. This has saved my blots many times, when I have had strong background.
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