western-quantitation??

Ian A. York iayork at panix.com
Wed Aug 26 09:42:13 EST 1998


In article <35e40dba.2700387 at news.univie.ac.at>,
Martin Offterdinger <a8803349.nospam at unet.univie.ac.at> wrote:
>
>I would like to know if you would consider it as science to quantitate
>bands obtained by enzymatic Western Blotting with a densitometer. I

I have seen this done, but only as "semi-quantitative", not
"quantitative".  As with any other method, you need to take great care
that your antibody isn't limiting and so forth.  One way of making the
data a little more convincing is to do serial dilutions of your lysate,
say 3-fold.  Your densitometry should accurately detect the known dilution
of each sample, and the ratios between the different samples should remain
constant at the different dilutions.  If either is not true, your blots
are not useful for quantitation.

My own limited experience in trying to get accurate quantitation from
westerns is that it's not very reliable, but that if you emphasize the
"semi" part of the semi-quantitative you can get some information from it.

It depends heavily on your protein and antibody, I imagine.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England



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