Introduction to Practical Biochemistry

György Hegyi

József Kardos

Mihály Kovács

András Málnási-Csizmadia

László Nyitray

Gábor Pál

László Radnai

Attila Reményi

István Venekei

This book is freely available for research and educational purposes. Reproduction in any form is prohibited without written permission of the owner.

Made in the project entitled "E-learning scientific content development in ELTE TTK" with number TÁMOP-4.1.2.A/1-11/1-2011-0073. Consortium leader: Eötvös Loránd University, Consortium Members: ELTE Faculties of Science Student Foundation, ITStudy Hungary Ltd.

Table of Contents

1. Common laboratory tools and equipment used in biochemistry and molecular biology
1.1. Biological samples and chemical substances in the laboratory
1.2. Plastic and glass tubes used for the storage of liquids
1.3. Beakers and laboratory flasks
1.4. Precise volumetric measurements with graduated cylinders and micropipettes
1.5. Mixing of liquids
1.6. Laboratory balances
1.7. Methods of sterilisation and in-house production of high-purity water
1.8. Working with cell cultures
1.9. Centrifuges
1.10. Other widely used laboratory techniques: spectrophotometry, electrophoresis, chromatography
1.11. Storage of biological samples
2. Units, solutions, dialysis
2.1. About units
2.2. Numeric expression of quantities
2.2.1. The accuracy of numbers, significant figures
2.2.2. Expression of large and small quantities: exponential and prefix forms
2.3. About solutions
2.3.1. Definition of solutions and their main characteristics
2.3.2. Quantitative description of solutions, concentration units
2.3.3. Preparation of solutions
2.4. Dialysis
2.4.1. The principle of dialysis
2.4.2. Practical aspects and applications of dialysis
3. Acid-base equilibria, pH, buffer systems
3.1. Ionisation equilibria of acids and bases in aqueous solutions
3.2. pH-stabilising acid-base systems (buffers) and the influence of pH on ionisation
3.3. Measurement of the pH
3.4. Demo calculations of charge and pI
3.4.1. Demonstration that pI is the average of the pKa values of the carboxylic acid and amino groups of an amino acid lacking an ionisable group in its side chain
3.4.2. Demonstration that the pI value of aspartic acid is the average of the pKa values of the two carboxylic acid groups in it
3.4.3. Demo calculation of the isoelectric point of a protein
4. Spectrophotometry and protein concentration measurements
4.1. Photometry
4.2. The UV-VIS photometer
4.3. Other possible uses of photometry
4.4. Frequently arising problems in photometry
4.5. Determination of protein concentration
4.5.1. Biuret test
4.5.2. Lowry (Folin) protein assay
4.5.3. Bradford protein assay
4.5.4. Spectrophotometry based on UV absorption
4.6. Spectrophotometry in practice: some examples
4.6.1. Absorption spectrum of ATP
4.6.2. Hyperchromicity of DNA
4.6.3. Absorption spectra and molecular structure of NAD and NADH
4.6.4. Absorption spectrum of proteins
4.6.5. Determination of the purity of DNA and protein samples
4.7. Fluorimetry
4.7.1. Physical basis of fluorescence
4.7.2. The fluorimeter
4.7.3. Fluorophores
4.8. Appendix
4.8.1. Fluorescence, phosphorescence and chemiluminescence
4.8.2. Photobleaching
4.8.3. Fluorescence anisotropy and circular dichroism
4.8.4. Quenching and FRET
5. Cell disruption, cell fractionation and protein isolation
5.1. Cell disruption
5.2. Cell fractionation
5.3. Centrifugation
5.3.1. Differential centrifugation: cell fractionation based primarily on particle size
5.3.2. Equilibrium density-gradient centrifugation: fractionation based on density
5.4. Low-resolution, large-scale protein fractionation
5.4.1. Fractionation methods based on solubility
5.4.2. Protein fractionation based on particle size
5.5. Lyophilisation (freeze-drying)
6. Chromatographic methods
6.1. Gel filtration chromatography
6.2. Ion exchange chromatography
6.3. Hydrophobic interaction chromatography
6.4. Affinity chromatography
6.5. High performance (high pressure) liquid chromatography (HPLC)
7. Electrophoresis
7.1. Principles of electrophoresis
7.2. About gel electrophoresis
7.3. Polyacrylamide gel electrophoresis (PAGE)
7.3.1. About the PAGE method in general
7.3.2. Native PAGE
7.3.3. SDS-PAGE
7.3.4. Isoelectric focusing
7.3.5. Two-dimensional (2D) electrophoresis
7.4. Agarose gel electrophoresis
7.5. Staining methods
7.5.1. General protein gel stains
7.5.2. General DNA gel stains
7.5.3. Specific protein detection methods: Western blot
7.5.4. Specific protein detection methods: In-gel method based on enzyme activity
7.6. Typical examples of protein-separating gel electrophoresis
7.6.1. Native PAGE separation and detection of lactate dehydrogenase isoenzymes
7.6.2. Molecular mass determination of myofibrillar proteins using SDS-PAGE
8. Protein-ligand interactions
8.1. Biomolecular interactions
8.2. Reaction kinetics
8.3. Protein-ligand interactions
8.4. Relationship between the free enthalpy (Gibbs free energy) change and the equilibrium constant
8.5. Molecular forces stabilising ligand binding
8.6. Determination of the binding constant
8.7. Methods for the experimental determination of the binding constant
8.7.1. Surface plasmon resonance (SPR)
8.7.2. Isothermal titration calorimetry (ITC)
8.7.3. Fluorescence depolarisation to characterise protein-ligand binding interactions
8.8. Test questions and problems
9. Enzyme kinetics
9.1 Thermodynamic interpretation of enzyme catalysis
9.2. Michaelis-Menten kinetics
9.3 Determination of initial reaction rates and principal kinetic parameters
9.4. Enzyme inhibition mechanisms
9.4.1. Competitive inhibition
9.4.2. Uncompetitive inhibition
9.4.3. Mixed inhibition
10. Recombinant DNA technology
10.1. Recombinant DNA techniques and molecular cloning
10.2. Plasmid vectors
10.3. Creation of recombinant DNA constructs
10.4. Introduction of recombinant DNA constructs into host cells and the identification of recombinant colonies
10.5. Isolation of plasmid DNA
10.6. Analysis of plasmid DNA by gel electrophoresis
10.7. Polymerase chain reaction (PCR)
10.8. Site directed in vitro mutagenesis
10.9. DNA sequencing
11. Bioinformatics
11.1. Introduction
11.2. Primary sequence and three-dimensional structure databases
11.2.1. GenBank
11.2.2. UniProt
11.2.3. Protein Data Bank (PDB)
11.3. Introduction to bioinformatics analysis of sequences
11.3.1. Bioinformatics tasks during molecular cloning
11.3.2. Sequence similarity search and sequence alignment
11.3.3. Bioinformatics analysis of protein sequences
11.4. Visualisation of protein structures by molecular graphics programs
11.4.1. RasMol
11.4.2. PyMOL
11.4.3. Jmol
12. Calculations and problem solving exercises
12.1. Useful preliminary information
12.2. Problems and exercises
12.2.1 Units of measure, solutions
12.2.2. Ionisation equilibria
12.2.3. Spectrophotometry of biomolecules
12.2.4. Cell disruption, cell fractionation and protein isolation
12.2.5. Peptides and proteins
12.2.6. Chromatographic methods
12.2.7. Electrophoretic methods
12.2.8. Protein-ligand interactions
12.2.9. Enzyme kinetics
12.2.10. Recombinant DNA technology
12.2.11. Bioinformatics
12.3. Solutions
12.3.1. Units of measure, solutions
12.3.2. Ionisation equilibria
12.3.3. Spectrophotometry of biomolecules
12.3.4. Cell disruption, cell fractionation and protein isolation
12.3.5. Peptides and proteins
12.3.6. Chromatographic methods
12.3.7. Electrophoretic methods
12.3.8. Protein-ligand interactions
12.3.9. Enzyme kinetics
12.3.10. Recombinant DNA technology
12.3.11. Bioinformatics
13. Epilogue