Quantification of Western Blot Signals on ECL Film

[IanMc]

What you are actually measuring with the scanner is the intensity of the 
transmitted light rather than OD.
This is indeed related to OD via a log relationship. Also AFAIK X-ray film 
only has a two-log range so isn't ideal for quantitative assays where there 
are large fold differences. If you have one of the newer densitometers that 
can image chemiluminescence directly that might give you a better dymanic 
range.

Regarding the spot sizes, I have always assumed that this is due to a 
combination of 1) some of the protein moving laterally along the blot by 
capillary action before it is bound to the blot and 2) you are measuring 
light incidence on the x-ray film and light travels in straight lines and 
may also be reflected so you can expect a small amount of spill over from 
the spots when the light intensity is high. Generally I check the peak 
shapes for flat tops or dips which indicate that the film has been 
overexposed.

Ian Mc


"WS" <novalidaddress from nurfuerspam.de> wrote in message 
news:1176243483.769601.125520 from b75g2000hsg.googlegroups.com...
> 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
>