protein methods - Studying Proteins in the Lab How do you...

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Studying Proteins in the Lab • How do you purify proteins? • How do you know what protein you have? • How do you study proteins in live cells?
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Learning Objectives Define the term assay Describe the basic steps in protein purification Relate different techniques used in protein purification to the physical or chemical properties of the protein Describe the basis for fractionating proteins using various chromatographic techniques (i.e. ion exchange, gel filtration, and affinity chromatography). Discuss various methods for detecting and identifying proteins in the lab (UV-light spectra, electrophoresis, Western blot, mass spectrometry). When is each one used? Why would you chose one over another? Describe how the following reagents are used in protein identification: sodium dodecyl sulfate, β -mercaptoethanol, trypsin, chymotrypsin, cyanogen bromide, polyampholytes Relate a protein’s amino acid composition to its pI, explain how you could determine the pI experimentally. Explain why green fluorescent protein (GFP) is a useful tag for studying proteins in vivo. Include in your explanation why this protein is fluorescent and how you can detect this fluorescence in the lab. Study Aids: Read Chapter 3 and do problems 7, 10, 12, and 14 Complete “Protein Methods Problem Set” available on D2L
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Why study proteins in the lab? • We have identified hundreds of thousands of proteins • To learn how each one works, biochemists must purify them individually • Once you have a purified protein you can determine its structure and function under defined laboratory conditions How do we know the sequence and structure?
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To purify proteins you need: A way to follow your protein, often by looking for its activity using an assay To know where in the cell to find your protein A way to separate your protein from the other proteins in the cell To protect your protein from being denatured or degraded
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How are you going to find your protein? First pass:
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This note was uploaded on 05/06/2010 for the course BIOC 460 taught by Professor Ziegler during the Spring '07 term at University of Arizona- Tucson.

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protein methods - Studying Proteins in the Lab How do you...

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