Selective separation of a target biomolecule from a highly complex biological matrix is based on the use of an affinity ligand that has been immobilized on a stationary phase packed in a short column. The ligand can be specific so that the antibody binds to only one epitope, or a group type ligand such as nucleotide analogues or Protein A or Protein G that will capture a group of selected compounds. Thus affinity ligands may be selected to serve a specific purpose, such as when immobilized antibody is used for antigen purification, or conversely when immobilized antigen is used for antibody purification.
Affinity Chromatography on Renewable Microcolumn
3.3.1.
Separation of mouse IgG on Protein A from bovine serum albumin on Sepharose microcolumn.
Sample: IgG 2µg/µL, BSA 4.6µg/µL. Injected volume 75 µL. Mobile phase PBS (0.14MNaCl, 3.8mM KCl, EDTA 1mM, phosphate 18mM, pH=7.4. Eluant 1M HCl, volume 5µL. Sample on column flow rate 5mL/sec, elution flow rate 2µL/sec. Column capacity 20mg/mL, column volume 10µL. Flow cell volume 6µL, optical path 3mm.