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Using Ion Exchange Chromatography to Separate Proteins Biotechnology: Science for the New Millennium Lab 9c Ellyn Daugherty SMBCP, San Mateo CA www.BiotechEd.com www.SMBiotech.com www.emcp.com/biotech www.sargentwelch.com/biotech Using Chromatography to Study and Separate Molecules Paper Chromatography Paper chromatography. Molecules separate as they move up the paper. The distance that the molecules travel depends on their size and solubility in the solvent. Using Chromatography to Study and Separate Molecules Thin-Layer Chromatography Thin-layer chromatography. Molecules separate as they move through the silica gel. Thin-layer chromatography is used to separate small molecules, such as amino acids. Column Chromatography How a Column Works Column Chromatography Open or Gravity Column Column Chromatography Gel-Filtration (Size-Exclusion) Chromatography Gel Filtration Resin. When starting protein purification, technicians sometimes use a gel-filtration (size-exclusion) column first. They know the molecular weight of their protein, so they can often eliminate several contaminant proteins by a quick run through a sizing column. Ion-Exchange Chromatography Ion Exchange Resin. Resins are manufactured with ions attached. The ions present a certain degree of positive or negative charge, depending on the buffer pH. Affinity Chromatography Affinity Chromatography. Separating molecules based on shape is often done using antibody resin. Antibodies recognize only certain antigens and will bind those and pull them out of solution (fraction #3). Column Chromatography in Biomanufacturing Fast-Performance Liquid Chromatography (FPLC) Fast-Performance Liquid Chromatography. Pumps push the buffer or sample through tubing, into and through the column. As fractions come off the column, they are run through a spectrophotometer that determines the protein concentration of the sample. Column Chromatography in Biomanufacturing Fast-Performance Liquid Chromatography (FPLC) High-Performance Liquid Chromatography (HPLC) Greatly improved ability to separate, purify, identify, and qualify samples. Biotechnology Curricular Support www.BiotechEd.com for PPTS, Syllabi, Acitivities, Hints, Links, etc [email protected] www.emcp.com/biotech Cheryl Monson [email protected] for books/IRC help www.SargentWelch.com/biotech Amy Naum for materials/equipment support [email protected]
