Course Dates
To be Announced
This is an introductory-level course designed to acquaint participants with the wide range of modern techniques available for separating and purifying biomolecules. The fundamentals of each technique will be presented, including practical examples; however, sufficient theoretical background will be provided to enable the participants to understand how each technique functions. Emphasis will be placed on techniques used in protein isolation and purification-both from native and recombinant sources. The course will be particularly valuable to those who are beginning work in biochemistry and biotechnology laboratories and who wish to broaden their background in separation procedures used in biochemistry. "Biochemical Separations" may be useful for persons getting back into the laboratory after several years, for those between jobs, and for scientists whose job description now requires familiarity with separation techniques used in Downstream Processing.
Course Outline
INTRODUCTION TO THE PROPERTIES OF BIOMOLECULES
Chemical and physical properties of proteins, nucleic acids, carbohydrates, lipids, and other biomolecules.
General strategies for molecular separations will be introduced.
PREPARATIVE METHODS
Survey of batch methods (salting out, isoelectric recipitation, and batch adsorption), dialysis, filtration, ultrafiltration.
Selected methods used in enzyme purification will be presented in detail.
BASICS OF LIQUID CHROMATOGRAPHY OF BIOMOLECULES
Principles of liquid chromatography. Emphasis will be placed on modern methods of low pressure liquid chromatography including gel filtration chromatography. Elementary chromatographic theory will be introduced.
ION EXCHANGE AND HYDROPHOBIC INTERACTION CHROTOGRAPHY
A survey of ion exchange and hydrophobic (reverse phase) separation methods for proteins, nucleic acids and smaller molecules. Examples will include both high pressure and low pressure methods.
AFFINITY CHROMATOGRAPHY
An introduction to this powerful method for purifying enzymes, antibodies, nucleic acids, and polysaccharides. Examples will include immobilized metal ion affinity chromatography, and affinity chromatography of enzymes with immobilized substrate analogues. Separation of recombinant proteins will be emphasized.
CONTEMPORARY PRACTICE OF LIQUID CHROMATOGRAPHY (HPLC)
An overview of the basic operating principles of HPLC with a comparison to other available tools. Special emphasis will be placed on the role of modem liquid chromatography as a problem-solving tool for bioanalytical research.
FUNDAMENTALS OF ELECTROPHORESIS
Basic principles of electrophoresis and survey of electrophoretic methods. Emphasis will be placed on polyacrylamide gel electrophoresis of proteins, molecular weight estimation by SDS PAGE, and isoelectric focusing
SEPARATION TECHNIQUES IN PROTEIN AND DNA SEQUENCING
Use of HPLC reverse phase chromatography in the sequencing of proteins by automated Edman degradation. Discussion of electrophoresis of nucleic acids as it applies to the sequencing of DNA.