western-quantitation??

Ian McFarlane i.mcfarlane at icrf.icnet.uk
Wed Aug 26 09:48:33 EST 1998


There is no reason why Western blots can not be scanned by a densitometer.
I have used this technique for ECL and AP blots. The bands are related to
the amount of protein present (cf ELISA). There are however several points
to be aware of.

1) When scanning you must ensure that you do not saturate the scanning
device ie that very black or white areas are not at the limit of the
dynamic range of the scanner. If the gradations between the signal and
background are weak I think it is acceptable to increase the sensitivity
of the scanner to compensate for this as long as the bands you are
comparing between are on the same blot.

2) With immunoblots the response of the system is not linear which
requires an appropiate standard curve to be included _on_each_blot_ so you
can control for deviations between blots.

3) Journals and reveiwers aren't keen on this sort of thing, don't ask me
why I don't know.

In short this method provides a useful method of comparing protein
expression between samples providing data on relative but not absolute
levels of protein expression.

Ian Mc






In article <35e40c48.2330687 at news.univie.ac.at>,
a8803349.nospam at unet.univie.ac.at (Martin Offterdinger) wrote:

> Hi!
> I would like to know if you would consider it as science to quantitate
> bands obtained by enzymatic Western Blotting with a densitometer. I
> would like to study the expression of a certain protein under the
> influence of hormones and compare it to control treated cell extracts.
> I know if you put the film under the densitometer you will get numbers
> (per cent of control) anyway. But is it good practice to conclude from
> the relative band intensity in such an experiment to the relative
> protein expression or is it just numbers that do not mean anything?
> I am looking forward to discussion.
> martin
> Martin Offterdinger
> Internal Med.I,Dept. Oncology
> University of Vienna
> Austria
> E-Mail:a8803349.nospam at unet.univie.ac.at
> (remove .nospam before mailing)



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