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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.

First steps--Develop an Assay An assay for an enzyme is a method for quantifying its activity. Since the assay is repeated many times, it is important that it be a simple procedure. Usually enzyme activity is monitored as a change in absorbance which can be measured using a spectrophotometer. For example an assay for ribonuclease measures the ...

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