Course

Practical

• Expression of concentration in terms of dilution, molarity, normality, percent expression
• pH measurement and buffer preparation
• Determination of absorption maxima of biomolecules
• Estimation of biomolecules through spectrophotometry and other methods
• Separation of carbohydrates and amino acids by paper chromatography
• Separation and analysis of fatty acids/lipids by GC
• Separation/estimation of biomolecules through HPLC and FPLC
• Separation of proteins using ion exchange, gel filtration and affinity chromatography
• Electrophoretic separation of proteins and nucleic acids
• Centrifugation- differential and density gradient
• (NH₄)₂SO₄ precipitation and dialysis
• Use of radioisotopes in metabolic studies
• PCR
• ELISA (Western blotting/ Dot blotting)

Unit I

Block 1: Separation Techniques

Principles and applications of separation techniques.

Unit I: Chromatography techniques (4 Lectures)

Principles and applications of paper, thin layer, gel filtration, ion-exchange, affinity, column & HPTLC, GC, HPLC and FPLC.

Unit II: Electrophoretic technique (2 Lectures)

General principles, paper and gel electrophoresis, native and SDS-PAGE, 2D-PAGE, capillary electrophoresis.

Unit III: Hydrodynamic methods (2 Lectures)

Hydrodyanmic methods of separation of biomolecules such as viscosity and sedimentation velocity, – their principles.

Unit IV: Centrifugation (2 Lectures)

Basic principles of sedimentation, type, care and safety aspects of centrifuge preparative and analytical centrifugation.

Unit II

Block 2: Spectroscopic Techniques

Unit I: Spectrophotometry (3 Lectures)

Principles and applications of UV-visible, Fluorescence, IR and FTIR, Raman, NMR and FTNMR, ESR and X-Ray spectroscopy.

Unit II: Mass spectroscopy (3 Lectures)

MS/MS, LC-MS, GC-MS, MALDI-TOF, applications of mass spectrometry in biochemistry.

Unit III: Atomic absorption spectrophotometry (2 Lectures)

Principle, function and instrumentation of atomic absorption spectrophotometry.

Unit III

Block 3. Microscopy

Unit I: Microscopic techniques (2 Lectures)

Principles and applications, light, UV, phase contrast, fluorescence and electron microscopy, flow cytometry.

Unit IV

Block 4: Tracer, Imaging, Immunochemical and Other Techniques

Unit I: Tracer technique (2 Lectures)

Tracer techniques in biology: concept of radioactivity, radioactivity counting methods with principles of different types of counters, concept of á, â and ã emitters, scintillation counters, J-ray spectrometers, autoradiography, applications of radioactive tracers in biology.

Unit II: Imaging techniques (2 Lectures)

Principles and applications of phosphor imager, MRI and CT scan.

Unit III: Immunochemical technique (2 Lectures)

Production of antibodies, immunoprecipitation, immunoblotting, immunoassays, RIA and ELISA.

Unit IV: Other techniques (2 Lectures)

Cryopreservation, polymerase chain reaction (PCR), FACS.

To provide hands-on experience to different biochemical techniques commonly used in research along with the knowledge on principles and the instrumentation.