Typical and often used methods for the preparation of biomolecules such as antigens and antibodies for cell research. Details are described in IMMUNO ENZYME TECHNIQUES IN CYTOCHEMISTRY as well as in the Cell Science chapters of this Homepage.
Principles of ion exchange chromatography, molecular sieving with gel filtration media and chromatofocusing. In the latter, the protein net charge determines its binding to the ion exchanger. In the course of chromatofocusing, a pH gradient from the column pH to eluant buffer pH is formed. The isoelectric point of the proteins and pH of the elution buffer determine protein migration.
Bioselective affinity chromatography is a most powerful separation technique. A biospecific ligand (f.e. antibody) is first attached to an insoluble matrix, and the molecules to be purified are selectively and reversibly absorbed to the immobilized ligand. Thus, antigens or antibodies may by coupled to agarose beads for the isolation of antibodies or antigens, respectively.