Protein Purification

The isolation of an individual polypeptide from a heterogeneous mix is an essential process in characterizing a protein of interest. In purified form a protein can be used to generate specific polyclonal and monoclonal antibodies for in vivo studies, in vitro the enzymic properties or interactions with nucleic acids or other proteins can be studied in detail and related to in viva function and, ultimately, the purified proteins can be used in structural determinations that define how polypeptide chains fold and amino acids interact to create a protein with a specific function. Protein purification exploits the properties a polypeptide derives from its unique amino acid composition and separation techniques rely on variations in solubility, size, charge, hydrophobicity and specific affinities to achieve fractionation. A combination of these methods is sufficient to isolate an individual protein from a complex mix. A prerequisite for any purification is the ability to unambiguously distinguish the protein of interest at all stages. This can be achieved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting with specific antisera, or by use of an assay specific for an activity of the protein.