densitometry on western blots

[martinhoehne At gmx.net]

Hello,

I have some basic questions on densitometry and would be glad if anyone
can point me to a good source to learn more or even answers some of the
questions.

1) As I understand it the bands have to be non-saturated. Does this
really mean that as soon as I have pixel values of 255 (on a 8-bit
image) I can forget about doing a densitometric analysis?

2) I want to use the analysis for judging the binding-strength of two
proteins. I have various point mutations of protein A and want to
compare how good these mutants interact with protein B. For this I am
doing a co-IP (precipitating protein A and co-precipitating protein B).
I then measure the intensities of the bands in western blots. In order
to not being fooled by slightly varying expression levels, I divide the
value for the band of the coprecipitated protein B by the value for the
precipitated protein protein A. Is this scientifically correct? Or is
there a better/more appropriate way?

Thanks for any input,

Martin