Production of Antisera Using Fusion Proteins

AbstractThe use of fusion proteins for the production of antisera allows specific areas of proteins to be targeted as epitopes and facilitates the purification of the antisera. This unit first describes the use of standard molecular biological techniques to construct a fusion‐protein expression plasmid by inserting a region of cDNA into a pGEX vector. Next, E. coli are transformed with the plasmid and induced to generate fusion protein. Also described is the purification of the soluble fusion protein, which is necessary for immunization and other subsequent procedures. This is accomplished by taking advantage of the GST fusion tag's affinity for glutathione. The purified fusion protein is then used to immunize animals, and antisera from these animals are then purified using affinity columns. Support protocols describe the construction and calibration of affinity columns for purifying antibodies using soluble fusion proteins, the use of insoluble fusion proteins for animal immunization, and preparation of affinity columns for purifying antibodies using insoluble fusion proteins.