Quantitating Westerns

Valerie Geffroy vgeffroy at ucdavis.edu
Fri Jun 30 16:18:35 EST 2000


Hi,
    The process of running a western is almost identical to the of ELISA. ie.
proteins are bound to a solid substrate, an antibody specific for the protein
of interest is conjugated to a reporter system, allowed to bind to the bound
protein and the reporter system activated. As with ELISA a standard curve is
constructed using a series of known concentrations of the protein of
interest. Provided you have a purified supply of your target, you can switch
to an alkaline phosphtase reporter system, run the western and locate the
region of your protein by staining one lane with BCIP and NBT. Line up the
lane with the unstained blot. Cut out the region of interest and place each
individual band in a test tube. React with PNPP and quantify in a
spectrophotometer at 405nm.
Establish a standard curve with your knowns and compare with your unknowns.
    I know this sounds like a lot of work, it really should take only an hour
or two after reacting with the antibody. Obviously there are many variations,
but they generally follow this same theme.

Jim Kami
Department of Agronomy  Hunt Hall Rm 272
University of California
1 Shields Avenue
Davis, CA 95616-8515 USA
email: jakami at ucdavis.edu
Tel: (530) 752-9982
Fax: (530) 752-4361

"Why does Common Sense always seem to be the least common sense ?!?"


Karthik Aghoram wrote:

> Hi,
>
> Is there a reliable way to quantitate protein expression from Western
> blots (am using a chemiluminescence system for band visualization)?  I
> have heard some phosphorimagers can also quantitate luminescent signals,
> but the one here (Mol Dynamics) does not.  Any ideas would be highly
> appreciated.
>
> Thank you very much.
> Karthik
>
> -------------------------------------------------------------------------
>
> Karthik Aghoram                         Postdoctoral Associate
>                                         Dept of Crop Science
> e-mail: kaghoram at unity.ncsu.edu         North Carolina St University
> Phone (W): 919-515-2705                 Raleigh, NC 27695-7620
>
> -------------------------------------------------------------------------
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