western-quantitation??

Ian McFarlane i.mcfarlane at icrf.icnet.uk
Wed Aug 26 10:26:13 EST 1998


In article <6s16s5$o92 at panix3.panix.com>, iayork at panix.com (Ian A. York) wrote:

> 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.

But if the response from the antibody is not linear then you will not be
able to predict the dilution factor.

What are the differences between an ELISA and a Western blot which lead to
one being quantitative and the other semi-q? 

Ian Mc



> 
> 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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