Western blots are one of the most widely used techniques in cell biology. In a western blot, investigators take advantage of the exquisite sensitivity of antibodies to identify proteins of interest in complex samples. In this lab, you will learn about the different kinds of antibodies used in western blots. You will use western blots to analyze Met and LacZ protein expression in your transformed yeast strains.
At the end of this lab, students will be able to:
Explain how monoclonal and polyclonal antibodies are produced.
Identify the different functional regions of antibodies and explain how they are used in western blots
Design a strategy that uses antibodies to detect epitope-tagged proteins.
Prepare a western blot to analyze protein expression in cell extracts.
Western blots provide a method to find the proverbial “needle in a haystack.” A typical cell expresses thousands of different proteins, and it is often difficult to detect changes in expression of your favorite protein (Yfp) without a probe that is capable of discriminating the Yfp against a large background of unrelated cellular proteins. Fortunately, antibodies provide highly specific molecular probes that can be used to detect the expression of proteins on western blots. To appreciate the sensitivity of western blots, it’s helpful to have some understanding of antibody structure and antibody production during immune respones. (Disclaimer: The following paragraphs provide a highly abbreviated overview of antibodies and one segment of the complex vertebrate immune system. The Department offers an immunology course that will introduce you to the finer details of this fascinating system.)