Unit 2998: Advanced Biochemical and Molecular Biology Techniques

1. Spectrophotometry

1.1 Principles of Spectrophotometry

• Interaction of light with matter
• Absorbance and transmittance definitions

1.2 Beer-Lambert Law

• Mathematical expression and variables
• Limitations and assumptions

1.3 Applications

• Measuring concentration of biomolecules (proteins, nucleic acids)
• Enzyme activity assays

2. Gel Electrophoresis

2.1 Fundamentals

• Principle: separation based on size and charge
• Types of gels: agarose vs polyacrylamide

2.2 Agarose Gel Electrophoresis

• Preparation and running conditions
• Applications for nucleic acids

2.3 Polyacrylamide Gel Electrophoresis (PAGE)

• SDS-PAGE for proteins
• Native PAGE
• Visualization techniques (staining, fluorescent dyes)

3. Enzyme Kinetics

3.1 Enzyme-Catalyzed Reaction Rates

• Definition of reaction rate
• Factors influencing rate

3.2 Michaelis-Menten Kinetics

• Derivation and significance of Km and Vmax
• Lineweaver-Burk and other plots

3.3 Enzyme Inhibition

• Competitive, non-competitive, and uncompetitive inhibition

3.4 Factors Affecting Enzyme Activity

• pH, temperature, substrate concentration

4. Protein Purification

4.1 Overview

• Importance and goals of purification
• Purity assessment

4.2 Chromatography Techniques

• Affinity chromatography: principle and applications
• Ion exchange chromatography: cation and anion exchange
• Size exclusion chromatography: separation by molecular size

4.3 Additional Methods

• Dialysis and ultrafiltration

5. Polymerase Chain Reaction (PCR)

5.1 Principles

• DNA amplification and the role of Taq polymerase
• Thermal cycling steps: denaturation, annealing, extension

5.2 Types of PCR

• Conventional PCR
• Quantitative PCR (qPCR): principles and applications
• Reverse transcription PCR (RT-PCR) overview

5.3 Applications

• Molecular diagnostics
• Genetic analysis

6. Western Blotting

6.1 Overview

• Purpose of western blotting

6.2 Procedure

• Protein separation by gel electrophoresis
• Transfer to membrane (nitrocellulose or PVDF)
• Blocking and antibody incubation
• Detection methods (chemiluminescence, fluorescence)

6.3 Data Analysis

• Interpretation of bands
• Quantification techniques

7. Mass Spectrometry

7.1 Principles

• Ionization methods (MALDI, ESI)
• Mass analyzers

7.2 Applications

• Protein identification and characterization
• Peptide sequencing
• Analysis of post-translational modifications

8. Cell Culture Techniques

8.1 Basics of Cell Culture

• Types of cell cultures: primary, immortalized, stem cells
• Sterile technique and aseptic handling

8.2 Cell Culture Media

• Components and preparation
• Serum and supplements

8.3 Cell Maintenance

• Passaging and subculturing
• Cell counting and viability assays
• Cryopreservation and thawing