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Why purify proteins? - UAB

11/10/06 Marilyn Niemann, UAB/CORD1 Why purify proteins? Detailed studies on function Determination of structure Industrial/pharmaceutical applications Generate antibodies Amino acid sequence determination1/10/06 Marilyn Niemann, UAB/CORD2 protein purification issues How much and how pure? Application Source Feasibility Native configuration? Functional/structural (yes) Microsequence (no) Antibody (maybe) Detection Method Functional assay Antigenic assay Band on gel21/10/06 Marilyn Niemann, UAB/CORD3 Affinitychromatographybinding to smallmoleculesIon exchangechromatographyIsoelectric point(charge)Size-exclusionchromatograph ySize / shapeMethodsPropertyTable of common methods of protein purificationsolubilityPrecipitation withammonium sulfate(salting out)**Ammonium sulfate precipitation is cheap, easy, and accommodates large sample is commonly one of the first steps in a purification scheme. Purification proceduresattempt to maintain theprotein in native some proteinscan be re-natured, mostcannot!

Solubility of Proteins • Solubility of a protein depends on ionic strength of the solution • Ionic strength is defined as I = (1/2) ∑ i C i Z i 2 • The sum is over all ionic species • Because the charge Zi is squared, divalent and trivalent ions contribute greatly to I. • Ci is the molar concentration of species i

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