Hoxhaj Gerta - Western blotting guru

1. Tables in Chapter 2

1. Figures in Chapter 1
2. Figures in Chapter 2
3. Figures in Chapter 3
4. Figures in Chapter 4
5. Figures in Chapter 6
6. Figures in Chapter 7

Ayaz Najafov

Harvard Medical School, Department of Cell Biology, Boston, MA, United States

Gerta Hoxhaj

Harvard T.H. Chan School of Public Health, Department of Genetics and Complex Diseases, Boston, MA, United States

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This book was written with an aim of providing biologists with a handy reference and guide for all aspects of performing Western blotting and obtaining consistent, reliable, high-quality data. We believe that this book will provide tremendous assistance for setting up Western blotting systems and solving problems associated with challenging cases. The intended target audience is both beginners and experts.

We did not delve in scrupulous details of the gel electrophoresis and immunoblotting theory. Instead, this book is a laboratory guide, and the emphasis here is on the technical aspects of employing Western blotting as a tool in molecular biology laboratories. The issues were described in bench terms, which makes the book more relevant to the end users of the technique.

General optimization and troubleshooting approaches and strategies, as well as a detailed guide for addressing specific Western blotting problems, tips and tricks developed and learned through years of experience with Western blotting, several special cases for Western blotting, and appendices with detailed protocols, as well as other useful information related to Western blotting, make this book a practical reference all molecular biology laboratories can benefit from.

The terms Western blotting and immunoblotting will be used interchangeably in the text. We will use the term target protein for the protein of interest to be analyzed by Western blotting. Room temperature will be routinely abbreviated as RT and molecular weight will be routinely abbreviated as MW.

All incubations for Western blotting membranes are to be done preferably on a side-to-side shaker/rocker. Radial shakers/rockers are not the top choice, since they tend to result in an uneven washing of the membranes.

Phospho-specific antibodies will be given primary consideration in the text. However, most of the discussed concepts can be applied to antibodies specific for other posttranslation modifications of proteins, such as methylation and acetylation.

Always wear appropriate personal protective equipment!

Note: Acrylamide is an acute toxin and a health hazard.

Note: Ponceau S is an irritant. Use necessary personal protective equipment.

Note: Acetic acid is flammable and corrosive.

This chapter introduces the reader to the origins of Western blotting, also known as immunoblotting. Western blotting is a popular technique among many disciplines of science. It allows the researcher to detect and quantify specific proteins using antibodies as probes. General information about this widely used technique and its applications are illustrated.