darker bands in WB - (Sep/28/2006 )

Hi all,

My WB looks good, defined bands and little background, but I´d like to have darker bands, I mean, "more black" and more intense. I´ve checked the expiry date of my ECL (Amersham) and it´s ok, and I´ve tried incubating more time with ECL and also increasing the time exposure, but nothing seems to work.
Can anyone help me?
thanks.

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load more antigens,

or increase the antibody concentration , but be careful to the background. In fact, if you already have a little background I would say that you have enough antibody.
you can also have a better signal by using an other primary antibody that would have more affinity for your antigen.
But honnestly, my mother always says that "better is the ennemy of good". It's already fine if you see bands. you might loose plenty of time trying to have darker bands.

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Ohh my boss always says that when we start to get muddled up trying to improve everything. It´s a good advice.
I will try another brand for the antibody but don´t lose time on this, and perhaps Photoshop may help a bit
Thanks

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more intense bands can be achieved by

optimize your antigen/Ab ratio (tedious)

longer exposition of blot to film (perhaps background also increase)

more sensitive ECL (if it must be GE/Amersham, ECLplus, or add some H2O2 to your ECL solution ~1 µl/5ml)

adapt your gamma value of your scanning software

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Thanks a lot

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I don't know how you develop your films, but I have started developing my films without a machine . And when I leave my films in the developer (liquid) for a longer time, the bands *obviously* get darker.

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I develop my films on a machine from the Radiology service (I work in a hospital), so I can´t control the times. I just introduce a cassete which has the membranes and the film inside, and the machine gives me the developed film.

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