MBB 222 Lecture Notes - Lecture 13: Column Chromatography, Ammonium Sulfate, Differential Centrifugation
3 Mar 2016
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Secreted: purify the protein from the culture supernatant. Lyse cells by mechanical means to obtain crude extract (osmotic shock, ultrasonic. Membrane bound or associated with a particular organelle: lyse cells and then fractionate them to purify the organelle or membrane fraction of interest. Differential centrifugation: centrifuge cell homogenate (i. e the soluble products of cell lysis) at increasing force to separate cells into their components. **in general, the smaller the subcellular component the greater the centrifugal force required to sediment it. Salting out : the solubility of proteins is generally lowered at high salt concentrations (selective aggregation or precipitation of proteins) Dialysis: separates proteins from small solutes (proteins are larger) The precipitate is then re-suspended in a physiological buffer. Proteins can be separated from small molecules by dialysis: Large molecules (like your protein) are retained in the dialysis bag. Small molecules are free to diffuse out into the dialysis buffer. Dialysis buffer is changed at least once.
