Western Blot Analysis

Western blot analysis separates proteins on the basis of size differences.

Whether naturally or artificially expressed, proteins are almost never found alone. Western blot analysis, or immunoblotting, is used for isolating and analyzing a protein of interest from a mixture.

Procedure

The application of voltage pulls proteins through a polymer gel at different rates.

Western blotting consists of three main steps: electrophoresis, in which the protein mixture is separated into localized bands based on size and charge; transfer, in which the proteins are moved from a polymer gel onto a paper substrate; and development, in which the protein band of interest is labeled and analyzed.

Applications in Research

A Western blot can help determine cell type.

Western blotting is extensively used in biomedical research. Because proteins are key indicators of cell function, a Western blot can provide information on cellular metabolism and lineage. Recombinant protein expression can also be verified and studied using a Western blot.

Applications in Medicine

A Western blot can help in the diagnosis of some prion diseases.

Western blots are labor intensive and time consuming, and their clinical applications are accordingly limited. However, they are important in diagnosing some diseases, including Lyme disease, HIV and prion diseases such as Creutzfeldt-Jakob Disease.

History

Because the polymer gel is not a good substrate for immunochemistry, efficient transfer was a key advance.

The first demonstration of protein separation in a polymer gel was published in 1977. The rapid and efficient transfer of the protein to paper by electroblotting--critical for the method's acceptance--was demonstrated in 1979.

Naming

As of 2010, there was no Eastern blot.

The term “Western blot” is part of a somewhat elaborate scientists' pun. The analogous procedure for characterizing DNA is called a Southern blot after its inventor, George Southern. The RNA version is universally known as a Northern blot and, perhaps inevitably, protein immunoblotting is Western blotting.

References

• “Recombinant DNA Technology”; O.S. Reddi; 2000
• “Applications and Engineering of Monoclonal Antibodies”; David J. King; 1998
• “Science in Medicine: The JCI Textbook of Molecular Medicine”; Andrew R. Marks, Ushma S. Neill; 2007
• “Basic Science Techniques in Clinical Practice”; Hitendra Patel, M. Arya, I. S. Shergill; 2007
• NINDS: Creutzfeldt-Jakob Disease Fact Sheet

Resources

• “A Guide to Methods in the Biomedical Sciences”; Ronald B. Corley; 2005
• “Principles and Reactions of Protein Extraction et seq...."; Hafiz Ahmed; 2005
• “Methods in Enzymology, Volume 449”; Lynne Maquat, Megerditch Kiledjian; 2008