MBB 222 Study Guide - Midterm Guide: Column Chromatography, Trichloroacetic Acid, Gene Expression
30 Nov 2017
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Protein must be overexpressed in bacteria, yeast, eukaryotic cells. Crude protein is first isolated in the cell fraction. Cytoplasmic proteins: cells must be lysed (ultrasonic vibration, homogenization, extrusion, osmotic shock, membrane solubilization) releasing the contents. Membrane-bound proteins or w/ organelle: fractionate the cells and purify the fraction of interest. Cells are lysed, making cell homogenate (products of cell lysis) Centrifuged at increasing force to separate cells into components (called differential centrifugation) The smaller the component, the more force is necessary to sediment it. Each fraction is tested for presence of desired protein. Can be purified from complex mixture by gradually increasing the concentration of salt. Salts, small proteins and other impurities can be removed. Large molecules are retained in the dialysis bag; small molecules diffuse out until equilibrium. Dialysis buffer is changed to dilute out unwanted molecules. Membranes can have different cut-offs to allow release of different sizes 1 dalton= 1g/mol (10kda= small)
