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notes for PROTEIN PURIFICATION - Ion – concentration of...

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Salting-out 101 Salt competes with protein for water. Eventually water binds with salt and proteins form hydrophobic bond with one another and precipitates. Precipitated protein are collected in the order of hydrophobicity. -solubility of protein -Hofmeister Series -temperature -pH (avoid pH at isoelectric point because it will precipitate the protein) Ion-exchange Anionic –negative protein Cationic – positive protein Gradients pH – change the charge of the protein to disrupt the binding to the stationary phase
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Unformatted text preview: Ion – concentration of salt Size exclusion Need the proteins to be very different in molecular weight. Must use different size polymers Repeatable Natural Binding Affinity SDS-Page-Method of visualizing proteins-Separates proteins based on size Preparing sample-Mechanical breakdown of cell-Proteins to be analyzed is mixed with SDS-Addition of reducing agent-Addition of tracking dye Preparing the gel-more acrylamide -> more cross-linking in the polymer -> decreasing the size of pores...
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