Biochemistry 2280A Study Guide - Midterm Guide: Ion Exchange, Proteome, Electrophoresis
20 Sep 2016
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Document Summary
Outline the steps required to purify a protein. Cell extract (homogenate): the contents that come out of a cell. Initial fractionation: separates class of proteins we are interested in. Isolation of protein: through series of chromatography steps. Chromatography: use of different materials to separate individual components of a complete mixture into fractions. Fractions examined to determine which contains the protein of interest. Fraction of interest is subject to additional chromatography until desired protein is isolated. Ion exchange chromatography: ion exchange columns filled with positive or negative beads. Association between protein and matrix depends on ph. Beads: tiny and have small pores, big enough for some proteins to stick. Proteins that cannot enter beads: washed out of the column first. Allow for estimate of protein size to fraction the proteins. Affinity chromatography: affinity column covalently contains matrix that is covalently bonded to ligand that interacts with protein of interest. Protein binds to ligand as the solution is washed over the column.
