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.

LEARNING OBJECTIVES:

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.

REFERENCES:

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