Principles of Biochemistry (2+1)

5.3.2.3 Protein Visualization and Assessment of Purification

i. SDS-polyacrylamide gel electrophoresis

SDS-polyacrylamide gel electrophoresis is performed in the presence of SDS (sodium dodecyl sulfate) which binds to proteins giving them a large net negative charge. Since the charges of all proteins are fairly equal, this method separates them almost entirely based on size. SDS-PAGE is often used to test the purity of a protein after each step in a series. As unwanted proteins are gradually removed from the mixture, the number of bands visualized on the SDS-PAGE gel, is reduced, until there is only one band representing the desired protein.

ii. Immunoblotting

Immunoblotting is a protein visualization technique applied in combination with affinity chromatography. Antibodies for a specific protein are used as ligands on an affinity chromatography column. The target protein is retained on the column, then removed by rinsing the column with a salt solution or other agents. Antibodies linked to radioactive or dye labels aid in detection of the target protein once it is separated from the rest of the mixture.