densitometry on western blots

martinhoehne At gmx.net via methods%40net.bio.net (by martinhoehne At gmx.net)
Fri Nov 24 09:01:16 EST 2006


Thank you for your response. A few more 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?
>
> No, it does not. As long as there is a calibration curve and your
> signal is within reasonable dynamic response range, you are
> fine. If you don't have response calibrated, going to 24 bits or
> whatever will change nothing at all.


Reasonable dynamic range - is the what one of the other posters said
just a few saturated pixels?
I read somewhere that saturation does not matter too much since the
bands are not only getting darker but also the area covered by the band
is getting larger, and this can be used as a measurement as well. But
this seems to be an exotic (if not wrong) statement to me.



> In order to deal quantitatively with non-linear processes in the course
> western/densitometry, you *have to have* a calibration curve that tells
> you how your signal changes quantitatively with changing input.
> If this can be done with purified protein, you then have a chance
> to express results in true protein quantity. In most cases the relative
> numbers are perfectly acceptable too: Use your pre-PI material
> and do 1:2 or so serial dilutions that cover your entire range of
> response. When done in parallel with the sample (same gel, same
> development, etc) this guarantees that you can express your results
> quantitatively, in relative units as % of total.

would it be ok to run the lysates on a second gel in parallel? The gel
with the IPs is already full.
Excuse me if the question sounds stupid, but how exactly do I proceed
then? Please correct me if I´m wrong.

Let´s assume I load 1:2 serial dilutions of the lysates and stain for
protein B.
- I do a densitometric analysis (I use the gel analysis tool of
ImageJ).
- I plot in a graph the amount of input (e.g. 0,125x; 0,25x; 0,5x; 1x)
versus the value obtained from ImageJ for the given band.
- I do the densitometric analysis for the precipitates (for both
proteins, A and B). For each band I take the value I obtained from
ImageJ and look up in my graph to what amount of protein this value
corresponds. Let´s say for protein A_wt it´s 0,8x and the
coprecipitated protein B in this lane it´s 0,6x. And for protein
A_mut1 it´s 0,7x and for the coprecipitated protein it´s 0,9x.
- I calculate (0,6 / 0,8) / (0,6 / 0,8) =1 and (0,9 / 0,7) / (0,6 /
0,8)=1,7

Can I then state that protein A_mut1 precipitates 1,7x more protein B
as protein A_wt does?



And in addition some technical questions:
Is it ok to scan the blot with an ordinary scanner using 300dpi
greyscale or do I have to opt for more dpi or even have to use a
transillumination scanner? 

Thanks a lot for your input, 

Martin



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