MBB 222 Lecture Notes - Lecture 11: Ammonium Sulfate, Column Chromatography, Differential Centrifugation

• School: Simon Fraser University
• Department: Molec Biol & Biochem
• Course: MBB 222
• Professor: Irina Kovalyova

Document Summary

Protein purification is a necessary first step in determining the molecular structure and function of a protein. The source of proteins to be characterized can be tissue homogenates, mammalian tissue culture cells, or bacteria cultures that express (i. e. , synthesize) specific recombinant protein. The first step in protein purification is to prepare a cell extract. Starting from a cell suspension, the cells can be disrupted to release their contents by sonication (applying sound energy to agitate particles in a sample), shearing, or treatment with mild detergents. The method used to prepare the initial cell extract needs to be gentle enough to preserve protein function as determined by biochemical assays. Centrifugation separates macromolecules on the basis of their density, centrifugal force (multuples of g), and the centrifugation time. A centrifuge consists of a rotational motor, a refrigerated chamber to reduce heat generation during the spin, and a rotor connected to the motor that holds the balanced centrifuge tubes.