Quantification of Western Blot Signals on ECL Film

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Tue Apr 10 17:18:03 EST 2007


Dear Experts,

I want to quantificate signals on X-Ray films obtained by Western
Blot / ECL.
Actually, I just scanned the films with an ordinary flatbed scanner
(transparency mode, internally 10 bit), that gives me 8bit tiff
images. I use the Ctrl_1/2/3 sequence from ImageJ to generate density
graphs of each lane which then are integrated with the wand tool.

Two issues now make me think and I am looking for a good solution:

1) Strong signals yield spots on the film which probably are bigger
than the actual spot was on the gel. Longer exposure times make larger
spots)

2) I am worried about the meaning of the area under the curve data as
I have scanned a microplate of a colorimetric assay and compared the
OD values obtained by the photometer with the grey values of the
wells obtained by scanning. The relationship is not linear unless I
would plot the log of the grey values against the OD. That only would
make sense if one applied Lamber-Beers's law I/Io=10^(-ecd) on the
data where I is the grey value and ecd is the OD value from the
photometer.

I also read in a tutorial on a laser densitometer from Molecular
Devices that this machine would convert the densitometric values to
greyscales for storage using a logarithmic relation that would
resemble the equation mentioned above.

My goal is to compare the amounts of proteins on the blots. May I
assume (within all the limits the use of xray films has) that double
the amount of protein yields double the area under the curve or is
the reality (even under the constraints mentioned) more complex. Is
there a reason (or even necessity) to use the log of the area in
order to obtain correct values?

Thanks for your help!

Wo



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