ABSTRACT

Principles of protein purification The basic aim in protein purification is to isolate one particular protein of interest from other contaminating proteins so that its structure and/or other properties can be studied. Once a suitable cellular source of the protein has been identified, the protein is liberated into solution and then separated from contaminating material by sequential use of a series of different fractionation techniques or separations. These separations exploit one or more of the following basic properties of the protein: its solubility, its size, its charge, its hydrophobicity or its specific binding affinity. These separations may be chromatographic techniques such as ion exchange, gel filtration or affinity chromatography, hydrophobic interaction chromatography, in which the protein binds to a hydrophobic material, or electrophoretic techniques such as isoelectric focusing (Section C2). Other electrophoretic procedures, mainly sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (Section C2), are used to monitor the extent of purification and to determine the molecular mass and subunit composition of the purified protein.