Distance Learning: Techniques in Structural Molecular Biology (TSMB)

This section provides an appreciation of the methodology for protein purification and crystallisation for structural studies.

Principles of Chromatography

Functional biomolecules and stability
Introduction to the dynamics of chromatography
Ion exchange chromatography
Gel filtration chromatography
Affinity chromatography
Immobilised metal affinity chromatography
Reverse phase chromatography
Hydrophobic interaction chromatography
High pressure liquid chromatography
Fast protein liquid chromatography
Polyacrylamide gel electrophoresis

Crystallisation of proteins

Introduction: Physical Principles
Sample Preparation
Protein Purity
Protein Concentration
Phase Diagram
Crystallisation Techniques
Batch
Microbatch
Vapour Diffusion by Hanging or Sitting Drop
Seeding
Equilibrium Dialysis
Gels
Automated Methods
Biochemical and Biophysical Crystallisation Parameters
Type of Precipitant
Concentration of Precipitant and Protein
Drop Volume and Make-up
Well Volume and Make-up
pH and Ionic Strength
Buffer
Additives
Temperature and Gravity
Crystallisation Screening and Optimisation
Grid Screening
Incomplete Factorial Design Screening
Optimisation

Objectives

To teach the basic principles of purification and crystallisation of proteins, including a) the biochemical techniques that can be applied to utilise the various chemical features and biological properties exhibited by these complex molecules and b) the crystallisation methodologies that can lead to the successful X-ray analysis of the three dimensional structure. At the end of this section, students should:

• Understand the different aspects of the chemical nature of a protein and how these can be used in purification
• Know the various principles involved in protein chromatography
• Be able to relate these principles to purification of protein suitable for crystallisation trials
• Understand the underlying principles of the different approaches adopted in crystallisation trials