Course

Unit 1

Protein extraction and quantitation: Enzymatic lysis, Homogenizer, Blender, Sonication, Bead mill shaker, French press, Biuret, Lowry, BCA and Bradford Assays.

Protein precipitation and treatment: Salting-in, Salting-out, Effect of organic solvents and polymers, Dialysis, Ultrafiltration, Centrifugation.

Unit 2

Chromatography: Partition coefficient, Retention, Resolution, Gel flitration chromatography, Ion exchange chromatography, Affinity chromatography, Hydrophobic interaction chromatography, Hydroxyapatite chromatography, Paper chromatography, Thin layer chromatography, Reversed-phase chromatography, Normal phase chromatography.

Unit 3

HPLC: Fundamentals of high-performance liquid chromatography, Columns, Detectors.

Unit 4

Electrophoresis: Native PAGE, SDS-PAGE, Isoelectric focusing, 2D-PAGE.

Unit 5

Spectroscopy: Fundamentals of UV/Vis Spectroscopy, Applications of UV/Vis spectroscopy, Spectrophotometer, Fundamentals of fluorescence spectroscopy, Jablonski diagram, Spectro fluorometer, Applications of spectrofluorimetry.

The main objective of this course is to provide basic knowledge to students to understand analytical tools and apply them to decipher structure and functions of biomolecules.

COURSE OUTCOMES:

After completing the course, students shall be able to

CO1. Describe important biomolecular extraction, quantitation, separation and purification techniques.

CO2. Recall concepts and applications of UV/Visible and fluorescence spectroscopy.

CO3. Differentiate important techniques to analyze biomolecules.

CO4. Solve qualitative and quantitative problems related to bio molecular characterization

• Protein Purification Techniques: A Practical Approach, Simon Roe, Oxford, 2nd Ed., 2004.
• Protein Purification: Principle and Practice, Robert K. Scopes, Springer, 3rd Ed., 1994.
• Physical Biochemistry: Principles and Applications, David Sheehan, John Wiley & Sons, 2nd Ed., 2000.
• Practical Biochemistry: Principles and Techniques, Keith Wilson and John Walker, Cambridge, 5th Ed., 2004.