quantification western blot

[ana martins]

Hello everyone,

I was wondering if you could give me some quick help regarding western blot
quantification with Image J. I read the tutorial which describes how to
quantify western blot bands.

What happens is that my membranes do not have a clean and uniform
background. Sometimes in the upper part of my interest band the background
is clean, whereas just in the lower part of my interest band I have some
strong background. So when I plot gel lanes I wont have the perfect example
of a straight line delimiting the background intensity following a nice
pick. So I was wondering how do you actually set the cut offs in this case?
Is there a more accurate way to subtract the background? I am concerned
because this way of doing it really affects my data and I am always in
doubt where to set the cut off when I don´t have a perfect background
intensity.


Chrees!

Sofia

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[Artsi]

Hi all!
Thank you Sofia for bringing this up, i wish someone here picks the glove
to give a good answer for this one, i have some points to add.
Western is a rather crude method on my humble opinion differences of less
then 20% are undetectable - basically if you can't see it and you have to
make a quantification it's not there.
When scanning one has to make sure the blot is not overexposed, i've
noticed that the best exposure to scan is one before the one which looks
best to the eye, digital cameras can show overexposure (is it any good?).
I think that in the case Sofia describes if the above the band the
background is white and below it's still dark one can draw an inclined line
at the bottom of the peak to account for the background.
And last i think that once upon a time people used to quantify gels with
photoshop - measuring the area of the band and average intensity how does
that relate to the peak (width is area height intensity).

Hanna

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