This webpage was created as an assignment for an undergraduate course at Davidson College.

The Western Blot

The Western blot, commonly called the protein blot, is a method used to separate proteins by molecular weight (MW in Da or kDa). Similar to the Southern blot for DNA and the Northern blot for RNA, the Western blot utilizes separation of the studied molecule through a medium. Among its various uses in the labratory, the Western blot has recently become a notable molecular tool because of its role in the ELISA test for detection of HIV. The most common Western blot method uses sodium dodecyl sulfate as a denaturing agent and polyacrylamide gel electrophoresis as the medium for separation (SDS-PAGE). Like Southern and Northern blotting, Western blotting shares several fundamental steps: extraction, denaturation, separation, transfer, blocking, hybridization with a probe, washing, viewing and analysis.




Figure 1. Depiction of SDS-PAGE setup utilizing a method that combines separation and transfer. (Image - Short Protocols in Molecular Biology, Second Edition, p. 10-34)



Figure 2. Nitrocellulose transfer via capillary action. (Highly sophisticated paper towel method). Permission pending from source.





Figure 3. Depicts detection by enzymatic Ab/protein hybrid via method developed by Amersham.
As the membrane is treated with peracid, the secondary Ab probe reacts enzymatically and produces light as a byproduct. The light produced is detected by film, which is then exposed for analysis. (Image - Basic Protein and Peptide Protocols, Vol 32 , p.235)


Figure 4. Top image: Film exposed after staining with SYPRO Ruby protein blot stain. Bottom image: Film after enzymatic action and exposure. Permission pending from source.


The Resolving Power of SDS-PAGE:

Figure 5. The resolving accuracy of MW in a typical SDS-PAGE Western blot. Note the complex MW standard in lane 8. Permission pending from source.


Useful Links:

See a real protocol for the Western Blot by Kirkegaard and Perry Laboratories here.

Buy materials to run your own Western blot here.

Try a common Western blot analysis problem here.


Ausubel, Frederick M. et. al., Short Protocols in Molecular Biology Second Edition. John Wiley and Sons: New York, 1997.

p. 10-33 - 10-35.


Walker, John M. ed. Basic Protein and Peptide Protocols Vol. 32, Humana Press: Totowa, NY, 1994. 189, 209-221.


White, Brian. Southerns, Northerns, Westerns, & Cloning: Molecular Searching Techniques.1995. February 17, 2003

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Campbell, Malcolm. Western Blot. 2001. February 16, 2003

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Biology At Davidson

Molecular Biology At Davidson

© Copyright 2003 Department of Biology, Davidson College, Davidson, NC 28036

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