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BIOC23: Lec 6 - Salting in and protein quantification.doc

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University of Toronto Scarborough
Biological Sciences
Rongmin Zhao

Lec 6 Salting inout and protein quantitationIf you change pH or include some organic mocs in solution the solubility of the protein sig changesCan still analyze solubility of protein even if denaturesSalting out can preserve protein activity but not necessarilyBy changing pH of buffer can precipitate protein out bc all proteins are made of AAs which are chargedSelective precipitation byopH variationswhen pH of buffer isnt equal to pI of protein then the protein has a net chargeproteins w same charge repel each otherwhen PHPI proteins dont have NET charge and they tend to aggregateso if change pH of solution can change solubility of solution and can precipitate the protein outoorganic solvesEtOH MeOH AcetoneAcetonePrecipitate protein but may harbor lipidschlorophyll which may dissolve in acetoneGood method to precipitate protein from small organic mocsChloroformEffective separate protein even from DNAMolecular crowding agentPEGAcids TCAquickly kills cell and precipitates protein outEven small amts can be precipitated outProtein becomes white when denaturedSpin down and collectSince strong acid after collecting precipitate must neutralize protein using NaOH or high concentration of Tris bufferPrinciple organic solvents DENATURE proteins exposing more hydrophobic sidesreduces proteins solubilityproteins aggregateCan use centrifugationCant detect enzyme activity bc denaturesoIonic strengthSALTING OUTSalting OutUsu with ammonium sulfatepreserves enzymatic activityMost proteins are less soluble at high salt concentraitonsThe salt concentration at which a protein precipitates differs from one protein to another bc diff solubilities at diff calt concentration
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