Bioanalytics Analytical Methods and Concepts in Biochemistry and Molecular Biology

Bioanalytics: Analytical Methods and Concepts in Biochemistry and Molecular Biology
Table of Contents
Preface
Introduction: Bioanalytics - a Science in its Own Right
Part I: Protein Analytics
	Chapter 1: Protein Purification
		1.1 Properties of Proteins
		1.2 Protein Localization and Purification Strategy
		1.3 Homogenization and Cell Disruption
		1.4 Precipitation
		1.5 Centrifugation
			1.5.1 Basic Principles
			1.5.2 Centrifugation Techniques
		1.6 Removal of Salts and Hydrophilic Contaminants
		1.7 Concentration
		1.8 Detergents and their Removal
			1.8.1 Properties of Detergents
			1.8.2 Removal of Detergents
		1.9 Sample Preparation for Proteome Analysis
		Further Reading
	Chapter 2: Protein determination
		2.1 Quantitative Determination by Staining Tests
			2.1.1 Biuret Assay
			2.1.2 Lowry Assay
			2.1.3 Bicinchoninic Acid Assay (BCA Assay)
			2.1.4 Bradford Assay
		2.2 Spectroscopic Methods
			2.2.1 Measurements in the UV Range
			2.2.2 Fluorescence Method
		2.3 Radioactive Labeling of Peptides and Proteins
			2.3.1 Iodinations
		Further Reading
	Chapter 3: Enzyme Activity Testing
		3.1 The Driving Force behind Chemical Reactions
		3.2 Rate of Chemical Reactions
		3.3 Catalysts
		3.4 Enzymes as Catalysts
		3.5 Rate of Enzyme-Controlled Reactions
		3.6 Michaelis-Menten Theory
		3.7 Determination of Km and Vmax
		3.8 Inhibitors
			3.8.1 Competitive Inhibitors
			3.8.2 Non-competitive Inhibitors
		3.9 Test System Set-up
			3.9.1 Analysis of the Physiological Function
			3.9.2 Selecting the Substrates
			3.9.3 Detection System
			3.9.4 Time Dependence
			3.9.5 pH Value
			3.9.6 Selecting the Buffer Substance and the Ionic Strength
			3.9.7 Temperature
			3.9.8 Substrate Concentration
			3.9.9 Controls
		Further Reading
	Chapter 4: Microcalorimetry
		4.1 Differential Scanning Calorimetry (DSC)
		4.2 Isothermal Titration Calorimetry (ITC)
			4.2.1 Ligand Binding to Proteins
			4.2.2 Binding of Molecules to Membranes: Insertion and Peripheral Binding
		4.3 Pressure Perturbation Calorimetry (PPC)
		Further Reading
	Chapter 5: Immunological Techniques
		5.1 Antibodies
			5.1.1 Antibodies and Immune Defense
			5.1.2 Antibodies as Reagents
			5.1.3 Properties of Antibodies
			5.1.4 Functional Structure of IgG
			5.1.5 Antigen Interaction at the Combining Site
			5.1.6 Handling of Antibodies
		5.2 Antigens
		5.3 Antigen-Antibody Reaction
			5.3.1 Immunoagglutination
			5.3.2 Immunoprecipitation
			5.3.3 Immune Binding
		5.4 Complement Fixation
		5.5 Methods in Cellular Immunology
		5.6 Alteration of Biological Functions
		5.7 Production of Antibodies
			5.7.1 Types of Antibodies
			5.7.2 New Antibody Techniques (Antibody Engineering)
			5.7.3 Optimized Monoclonal Antibody Constructs with Effector Functions for Therapeutic Application
		5.8 Outlook: Future Expansion of the Binding Concepts
		Dedication
		Further Reading
	Chapter 6: Chemical Modification of Proteins and Protein Complexes
		6.1 Chemical Modification of Protein Functional Groups
		6.2 Modification as a Means to Introduce Reporter Groups
			6.2.1 Investigation with Naturally Occurring Proteins
			6.2.2 Investigation of Recombinant and Mutated Proteins
		6.3 Protein Crosslinking for the Analysis of Protein Interaction
			6.3.1 Bifunctional Reagents
			6.3.2 Photoaffinity Labeling
		Further Reading
	Chapter 7: Spectroscopy
		7.1 Physical Principles and Measuring Techniques
			7.1.1 Physical Principles of Optical Spectroscopic Techniques
			7.1.2 Interaction of Light with Matter
			7.1.3 Absorption Measurement and the Lambert-Beer Law
			7.1.4 Photometer
			7.1.5 Time-Resolved Spectroscopy
		7.2 UV/VIS/NIR Spectroscopy
			7.2.1 Basic Principles
			7.2.2 Chromoproteins
		7.3 Fluorescence Spectroscopy
			7.3.1 Basic Principles of Fluorescence Spectroscopy
			7.3.2 Fluorescence: Emission and Action Spectra
			7.3.3 Fluorescence Studies using Intrinsic and Extrinsic Probes
			7.3.4 Green Fluorescent Protein (GFP) as a Unique Fluorescent Probe
			7.3.5 Quantum Dots as Fluorescence Labels
			7.3.6 Special Fluorescence Techniques: FRAP, FLIM, FCS, TIRF
			7.3.7 Förster Resonance Energy Transfer (FRET)
			7.3.8 Frequent Mistakes in Fluorescence Spectroscopy: ``The Seven Sins of Fluorescence Measurements´´
		7.4 Infrared Spectroscopy
			7.4.1 Basic Principles of IR Spectroscopy
			7.4.2 Molecular Vibrations
			7.4.3 Technical aspects of Infrared Spectroscopy
			7.4.4 Infrared Spectra of Proteins
		7.5 Raman Spectroscopy
			7.5.1 Basic Principles of Raman Spectroscopy
			7.5.2 Raman Experiments
			7.5.3 Resonance Raman Spectroscopy
		7.6 Single Molecule Spectroscopy
		7.7 Methods using Polarized Light
			7.7.1 Linear Dichroism
			7.7.2 Optical Rotation Dispersion and Circular Dichroism
		Further Reading
	Chapter 8: Light Microscopy Techniques - Imaging
		8.1 Steps on the Road to Microscopy - from Simple Lenses to High Resolution Microscopes
		8.2 Modern Applications
		8.3 Basic Physical Principles
		8.4 Detection Methods
		8.5 Sample Preparation
		8.6 Special Fluorescence Microscopic Analysis
		Further Reading
	Chapter 9: Cleavage of Proteins
		9.1 Proteolytic Enzymes
		9.2 Strategy
		9.3 Denaturation of Proteins
		9.4 Cleavage of Disulfide Bonds and Alkylation
		9.5 Enzymatic Fragmentation
			9.5.1 Proteases
			9.5.2 Conditions for Proteolysis
		9.6 Chemical Fragmentation
		9.7 Summary
		Further Reading
	Chapter 10: Chromatographic Separation Methods
		10.1 Instrumentation
		10.2 Fundamental Terms and Concepts in Chromatography
		10.3 Biophysical Properties of Peptides and Proteins
		10.4 Chromatographic Separation Modes for Peptides and Proteins
			10.4.1 High-Performance Size Exclusion Chromatography
			10.4.2 High-Performance Reversed-Phase Chromatography (HP-RPC)
			10.4.3 High-Performance Normal-Phase Chromatography (HP-NPC)
			10.4.4 High-Performance Hydrophilic Interaction Chromatography (HP-HILIC)
			10.4.5 High-Performance Aqueous Normal Phase Chromatography (HP-ANPC)
			10.4.6 High-Performance Hydrophobic Interaction Chromatography (HP-HIC)
			10.4.7 High-Performance Ion Exchange Chromatography (HP-IEX)
			10.4.8 High-Performance Affinity Chromatography (HP-AC)
		10.5 Method Development from Analytical to Preparative Scale Illustrated for HP-RPC
			10.5.1 Development of an Analytical Method
			10.5.2 Scaling up to Preparative Chromatography
			10.5.3 Fractionation
			10.5.4 Analysis of Fractionations
		10.6 Multidimensional HPLC
			10.6.1 Purification of Peptides and Proteins by MD-HPLC Methods
			10.6.2 Fractionation of Complex Peptide and Protein Mixtures by MD-HPLC
			10.6.3 Strategies for MD-HPLC Methods
			10.6.4 Design of an Effective MD-HPLC Scheme
		10.7 Final Remarks
		Further Reading
	Chapter 11: Electrophoretic Techniques
		11.1 Historical Review
		11.2 Theoretical Fundamentals
		11.3 Equipment and Procedures of Gel Electrophoreses
			11.3.1 Sample Preparation
			11.3.2 Gel Media for Electrophoresis
			11.3.3 Detection and Quantification of the Separated Proteins
			11.3.4 Zone Electrophoresis
			11.3.5 Porosity Gradient Gels
			11.3.6 Buffer Systems
			11.3.7 Disc Electrophoresis
			11.3.8 Acidic Native Electrophoresis
			11.3.9 SDS Polyacrylamide Gel Electrophoresis
			11.3.10 Cationic Detergent Electrophoresis
			11.3.11 Blue Native Polyacrylamide Gel Electrophoresis
			11.3.12 Isoelectric Focusing
		11.4 Preparative Techniques
			11.4.1 Electroelution from Gels
			11.4.2 Preparative Zone Electrophoresis
			11.4.3 Preparative Isoelectric Focusing
		11.5 Free Flow Electrophoresis
		11.6 High-Resolution Two-Dimensional Electrophoresis
			11.6.1 Sample Preparation
			11.6.2 Prefractionation
			11.6.3 First Dimension: IEF in IPG Strips
			11.6.4 Second Dimension: SDS Polyacrylamide Gel Electrophoresis
			11.6.5 Detection and Identification of Proteins
			11.6.6 Difference Gel Electrophoresis (DIGE)
		11.7 Electroblotting
			11.7.1 Blot Systems
			11.7.2 Transfer Buffers
			11.7.3 Blot Membranes
		Further Reading
	Chapter 12: Capillary Electrophoresis
		12.1 Historical Overview
		12.2 Capillary Electrophoresis Setup
		12.3 Basic Principles of Capillary Electrophoresis
			12.3.1 Sample Injection
			12.3.2 The Engine: Electroosmotic Flow (EOF)
			12.3.3 Joule Heating
			12.3.4 Detection Methods
		12.4 Capillary Electrophoresis Methods
			12.4.1 Capillary Zone Electrophoresis (CZE)
			12.4.2 Affinity Capillary Electrophoresis (ACE)
			12.4.3 Micellar Electrokinetic Chromatography (MEKC)
			12.4.4 Capillary Electrochromatography (CEC)
			12.4.5 Chiral Separations
			12.4.6 Capillary Gel Electrophoresis (CGE)
			12.4.7 Capillary Isoelectric Focusing (CIEF)
			12.4.8 Isotachophoresis (ITP)
		12.5 Special Techniques
			12.5.1 Sample Concentration
			12.5.2 Online Sample Concentration
			12.5.3 Fractionation
			12.5.4 Microchip Electrophoresis
		12.6 Outlook
		Further Reading
	Chapter 13: Amino Acid Analysis
		13.1 Sample Preparation
			13.1.1 Acidic Hydrolysis
			13.1.2 Alkaline Hydrolysis
			13.1.3 Enzymatic Hydrolysis
		13.2 Free Amino Acids
		13.3 Liquid Chromatography with Optical Detection Systems
			13.3.1 Post-Column Derivatization
			13.3.2 Pre-column Derivatization
		13.4 Amino Acid Analysis using Mass Spectrometry
		13.5 Summary
		Further Reading
	Chapter 14: Protein Sequence Analysis
		14.1 N-Terminal Sequence Analysis: The Edman Degradation
			14.1.1 Reactions of the Edman Degradation
			14.1.2 Identification of the Amino Acids
			14.1.3 Quality of Edman Degradation: the Repetitive Yield
			14.1.4 Instrumentation
			14.1.5 Problems of Amino Acid Sequence Analysis
			14.1.6 State of the Art
		14.2 C-Terminal Sequence Analysis
			14.2.1 Chemical Degradation Methods
			14.2.2 Peptide Quantities and Quality of the Chemical Degradation
			14.2.3 Degradation of Polypeptides with Carboxypeptidases
		Further Reading
	Chapter 15: Mass Spectrometry
		15.1 Ionization Methods
			15.1.1 Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)
			15.1.2 Electrospray Ionization (ESI)
		15.2 Mass Analyzer
			15.2.1 Time-of-Flight Analyzers (TOF)
			15.2.2 Quadrupole Analyzer
			15.2.3 Electric Ion Traps
			15.2.4 Magnetic Ion Trap
			15.2.5 Orbital Ion Trap
			15.2.6 Hybrid Instruments
		15.3 Ion Detectors
			15.3.1 Secondary Electron Multiplier (SEV)
			15.3.2 Faraday Cup
		15.4 Fragmentation Techniques
			15.4.1 Collision Induced Dissociation (CID)
			15.4.2 Prompt and Metastable Decay (ISD, PSD)
			15.4.3 Photon-Induced Dissociation (PID, IRMPD)
			15.4.4 Generation of Free Radicals (ECD, HECD, ETD)
		15.5 Mass Determination
			15.5.1 Calculation of Mass
			15.5.2 Influence of Isotopy
			15.5.3 Calibration
			15.5.4 Determination of the Number of Charges
			15.5.5 Signal Processing and Analysis
			15.5.6 Derivation of the Mass
			15.5.7 Problems
		15.6 Identification, Detection, and Structure Elucidation
			15.6.1 Identification
			15.6.2 Verification
			15.6.3 Structure Elucidation
		15.7 LC-MS and LC-MS/MS
			15.7.1 LC-MS
			15.7.2 LC-MS/MS
			15.7.3 Ion Mobility Spectrometry (IMS)
		15.8 Quantification
		Further Reading
	Chapter 16: Protein-Protein Interactions
		16.1 The Two-Hybrid System
			16.1.1 Principle of Two-Hybrid Systems
			16.1.2 Elements of the Two-Hybrid System
			16.1.3 Construction of Bait and Prey Proteins
			16.1.4 Which Bait Proteins can be used in a Y2H Screen?
			16.1.5 AD Fusion Proteins and cDNA Libraries
			16.1.6 Carrying out a Y2H Screen
			16.1.7 Other Modifications and Extensions of the Two-Hybrid-Technology
			16.1.8 Biochemical and Functional Analysis of Interactions
		16.2 TAP-Tagging and Purification of Protein Complexes
		16.3 Analyzing Interactions In Vitro: GST-Pulldown
		16.4 Co-immunoprecipitation
		16.5 Far-Western
		16.6 Surface Plasmon Resonance Spectroscopy
		16.7 Fluorescence Resonance Energy Transfer (FRET)
			16.7.1 Introduction
			16.7.2 Key Physical Principles of FRET
			16.7.3 Methods of FRET Measurements
			16.7.4 Fluorescent Probes for FRET
			16.7.5 Alternative Tools for Probing Protein-Protein Interactions: LINC and STET
		16.8 Analytical Ultracentrifugation
			16.8.1 Principles of Instrumentation
			16.8.2 Basics of Centrifugation
			16.8.3 Sedimentation Velocity Experiments
			16.8.4 Sedimentation-Diffusion Equilibrium Experiments
		Further Reading
	Chapter 17: Biosensors
		17.1 Dry Chemistry: Test Strips for Detecting and Monitoring Diabetes
		17.2 Biosensors
			17.2.1 Concept of Biosensors
			17.2.2 Construction and Function of Biosensors
			17.2.3 Cell Sensors
			17.2.4 Immunosensors
		17.3 Biomimetic Sensors
		17.4 From Glucose Enzyme Electrodes to Electronic DNA Biochips
		17.5 Resume: Biosensor or not Biosensor is no Longer the Question
		Further Reading
Part II: 3D Structure Determination
	Chapter 18. Magnetic Resonance Spectroscopy of Biomolecules
		18.1 NMR Spectroscopy of Biomolecules
			18.1.1 Theory of NMR Spectroscopy
			18.1.2 One-Dimensional NMR Spectroscopy
			18.1.3 Two-Dimensional NMR Spectroscopy
			18.1.4 Three-Dimensional NMR Spectroscopy
			18.1.5 Resonance Assignment
			18.1.6 Protein Structure Determination
			18.1.7 Protein Structures and more - an Overview
		18.2 EPR Spectroscopy of Biological Systems
			18.2.1 Basics of EPR Spectroscopy
			18.2.2 cw- EPR Spectroscopy
			18.2.3 g-Value
			18.2.4 Electron Spin Nuclear Spin Coupling (Hyperfine Coupling)
			18.2.5 g and Hyperfine Anisotropy
			18.2.6 Electron Spin-Electron Spin Coupling
			18.2.7 Pulsed EPR Experiments
			18.2.8 Further Examples of EPR Applications
			18.2.9 General Remarks on the Significance of EPR Spectra
			18.2.10 Comparison EPR/NMR
		Acknowledgements
		Further Reading
	Chapter 19: Electron Microscopy
		19.1 Transmission Electron Microscopy - Instrumentation
		19.2 Approaches to Preparation
			19.2.1 Native Samples in Ice
			19.2.2 Negative Staining
			19.2.3 Metal Coating by Evaporation
			19.2.4 Labeling of Proteins
		19.3 Imaging Process in the Electron Microscope
			19.3.1 Resolution of a Transmission Electron Microscope
			19.3.2 Interactions of the Electron Beam with the Object
			19.3.3 Phase Contrast in Transmission Electron Microscopy
			19.3.4 Electron Microscopy with a Phase Plate
			19.3.5 Imaging Procedure for Frozen-Hydrated Specimens
			19.3.6 Recording Images - Cameras and the Impact of Electrons
		19.4 Image Analysis and Processing of Electron Micrographs
			19.4.1 Pixel Size
			19.4.2 Fourier Transformation
			19.4.3 Analysis of the Contrast Transfer Function and Object Features
			19.4.4 Improving the Signal-to-Noise Ratio
			19.4.5 Principal Component Analysis and Classification
		19.5 Three-Dimensional Electron Microscopy
			19.5.1 Three-Dimensional Reconstruction of Single Particles
			19.5.2 Three-Dimensional Reconstruction of Regularly Arrayed Macromolecular Complexes
			19.5.3 Electron Tomography of Individual Objects
		19.6 Analysis of Complex 3D Data Sets
			19.6.1 Hybrid Approach: Combination of EM and X-Ray Data
			19.6.2 Segmenting Tomograms and Visualization
			19.6.3 Identifying Protein Complexes in Cellular Tomograms
		19.7 Perspectives of Electron Microscopy
		Further Reading
	Chapter 20: Atomic Force Microscopy
		20.1 Introduction
		20.2 Principle of the Atomic Force Microscope
		20.3 Interaction between Tip and Sample
		20.4 Preparation Procedures
		20.5 Mapping Biological Macromolecules
		20.6 Force Spectroscopy of Single Molecules
		20.7 Detection of Functional States and Interactions of Individual Proteins
		Further Reading
	Chapter 21: X-Ray Structure Analysis
		21.1 X-Ray Crystallography
			21.1.1 Crystallization
			21.1.2 Crystals and X-Ray Diffraction
			21.1.3 The Phase Problem
			21.1.4 Model Building and Structure Refinement
		21.2 Small Angle X-Ray Scattering (SAXS)
			21.2.1 Machine Setup
			21.2.2 Theory
			21.2.3 Data Analysis
		21.3 X-Ray Free Electron LASER (XFEL)
			21.3.1 Machine Setup and Theory
		Acknowledgement
		Further Reading
Part III: Peptides, Carbohydrates, and Lipids
	Chapter 22: Analytics of Synthetic Peptides
		22.1 Concept of Peptide Synthesis
		22.2 Purity of Synthetic Peptides
		22.3 Characterization and Identity of Synthetic Peptides
		22.4 Characterization of the Structure of Synthetic Peptides
		22.5 Analytics of Peptide Libraries
		Further Reading
	Chapter 23: Carbohydrate Analysis
		23.1 General Stereochemical Basics
			23.1.1 The Series of d-Sugars
			23.1.2 Stereochemistry of d-Glucose
			23.1.3 Important Monosaccharide Building Blocks
			23.1.4 The Series of l-Sugars
			23.1.5 The Glycosidic Bond
		23.2 Protein Glycosylation
			23.2.1 Structure of the N-Glycans
			23.2.2 Structure of the O-Glycans
		23.3 Analysis of Protein Glycosylation
			23.3.1 Analysis on the Basis of the Intact Glycoprotein
			23.3.2 Mass Spectrometric Analysis on the Basis  of Glycopeptides
			23.3.3 Release and Isolation of the N-Glycan Pool
			23.3.4 Analysis of Individual N-Glycans
		23.4 Genome, Proteome, Glycome
		23.5 Final Considerations
		Further Reading
	Chapter 24: Lipid Analysis
		24.1 Structure and Classification of Lipids
		24.2 Extraction of Lipids from Biological Sources
			24.2.1 Liquid Phase Extraction
			24.2.2 Solid Phase Extraction
		24.3 Methods for Lipid Analysis
			24.3.1 Chromatographic Methods
			24.3.2 Mass Spectrometry
			24.3.3 Immunoassays
			24.3.4 Further Methods in Lipid Analysis
			24.3.5 Combining Different Analytical Systems
		24.4 Analysis of Selected Lipid Classes
			24.4.1 Whole Lipid Extracts
			24.4.2 Fatty Acids
			24.4.3 Nonpolar Neutral Lipids
			24.4.4 Polar Ester Lipids
			24.4.5 Lipid Hormones and Intracellular Signaling Molecules
		24.5 Lipid Vitamins
		24.6 Lipidome Analysis
		24.7 Perspectives
		Further Reading
	Chapter 25: Analysis of Post-translational Modifications: Phosphorylation and Acetylation of Proteins
		25.1 Functional Relevance of Phosphorylation and Acetylation
			25.1.1 Phosphorylation
			25.1.2 Acetylation
		25.2 Strategies for the Analysis of Phosphorylated and Acetylated Proteins and Peptides
		25.3 Separation and Enrichment of Phosphorylated and Acetylated Proteins and Peptides
		25.4 Detection of Phosphorylated and Acetylated Proteins and Peptides
			25.4.1 Detection by Enzymatic, Radioactive, Immunochemical, and Fluorescence Based Methods
			25.4.2 Detection of Phosphorylated and Acetylated Proteins by Mass Spectrometry
		25.5 Localization and Identification of Post-translationally Modified Amino Acids
			25.5.1 Localization of Phosphorylated and Acetylated Amino Acids by Edman Degradation
			25.5.2 Localization of Phosphorylated and Acetylated Amino Acids by Tandem Mass Spectrometry
		25.6 Quantitative Analysis of Post-translational Modifications
		25.7 Future of Post-translational Modification Analysis
		Further Reading
Part IV: Nucleic Acid Analytics
	Chapter 26. Isolation and Purification of Nucleic Acids
		26.1 Purification and Determination of Nucleic Acid Concentration
			26.1.1 Phenolic Purification of Nucleic Acids
			26.1.2 Gel Filtration
			26.1.3 Precipitation of Nucleic Acids with Ethanol
			26.1.4 Determination of the Nucleic Acid Concentration
		26.2 Isolation of Genomic DNA
		26.3 Isolation of Low Molecular Weight DNA
			26.3.1 Isolation of Plasmid DNA from Bacteria
			26.3.2 Isolation of Eukaryotic Low Molecular Weight DNA
		26.4 Isolation of Viral DNA
			26.4.1 Isolation of Phage DNA
			26.4.2 Isolation of Eukaryotic Viral DNA
		26.5 Isolation of Single-Stranded DNA
			26.5.1 Isolation of M13 Phage DNA
			26.5.2 Separation of Single- and Double-Stranded DNA
		26.6 Isolation of RNA
			26.6.1 Isolation of Cytoplasmic RNA
			26.6.2 Isolation of Poly(A) RNA
			26.6.3 Isolation of Small RNA
		26.7 Isolation of Nucleic Acids using Magnetic Particles
		26.8 Lab-on-a-chip
		Further Reading
	Chapter 27: Analysis of Nucleic Acids
		27.1 Restriction Analysis
			27.1.1 Principle of Restriction Analyses
			27.1.2 Historical Overview
			27.1.3 Restriction Enzymes
			27.1.4 In Vitro Restriction and Applications
		27.2 Electrophoresis
			27.2.1 Gel Electrophoresis of DNA
			27.2.2 Gel Electrophoresis of RNA
			27.2.3 Pulsed-Field Gel Electrophoresis (PFGE)
			27.2.4 Two-Dimensional Gel Electrophoresis
			27.2.5 Capillary Gel Electrophoresis
		27.3 Staining Methods
			27.3.1 Fluorescent Dyes
			27.3.2 Silver Staining
		27.4 Nucleic Acid Blotting
			27.4.1 Nucleic Acid Blotting Methods
			27.4.2 Choice of Membrane
			27.4.3 Southern Blotting
			27.4.4 Northern Blotting
			27.4.5 Dot- and Slot-Blotting
			27.4.6 Colony and Plaque Hybridization
		27.5 Isolation of Nucleic Acid Fragments
			27.5.1 Purification using Glass Beads
			27.5.2 Purification using Gel Filtration or Reversed Phase
			27.5.3 Purification using Electroelution
			27.5.4 Other Methods
		27.6 LC-MS of Oligonucleotides
			27.6.1 Principles of the Synthesis of Oligonucleotides
			27.6.2 Investigation of the Purity and Characterization of Oligonucleotides
			27.6.3 Mass Spectrometric Investigation of Oligonucleotides
			27.6.4 IP-RP-HPLC-MS Investigation of a Phosphorothioate Oligonucleotide
		Further Reading
	Chapter 28: Techniques for the Hybridization and Detection of Nucleic Acids
		28.1 Basic Principles of Hybridization
			28.1.1 Principle and Practice of Hybridization
			28.1.2 Specificity of the Hybridization and Stringency
			28.1.3 Hybridization Methods
		28.2 Probes for Nucleic Acid Analysis
			28.2.1 DNA Probes
			28.2.2 RNA Probes
			28.2.3 PNA Probes
			28.2.4 LNA Probes
		28.3 Methods of Labeling
			28.3.1 Labeling Positions
			28.3.2 Enzymatic Labeling
			28.3.3 Photochemical Labeling Reactions
			28.3.4 Chemical Labeling
		28.4 Detection Systems
			28.4.1 Staining Methods
			28.4.2 Radioactive Systems
			28.4.3 Non-radioactive Systems
		28.5 Amplification Systems
			28.5.1 Target Amplification
			28.5.2 Target-Specific Signal Amplification
			28.5.3 Signal Amplification
		Further Reading
	Chapter 29: Polymerase Chain Reaction
		29.1 Possibilities of PCR
		29.2 Basics
			29.2.1 Instruments
			29.2.2 Amplification of DNA
			29.2.3 Amplification of RNA (RT-PCR)
			29.2.4 Optimizing the Reaction
			29.2.5 Quantitative PCR
		29.3 Special PCR Techniques
			29.3.1 Nested PCR
			29.3.2 Asymmetric PCR
			29.3.3 Use of Degenerate Primers
			29.3.4 Multiplex PCR
			29.3.5 Cycle sequencing
			29.3.6 In Vitro Mutagenesis
			29.3.7 Homogeneous PCR Detection Procedures
			29.3.8 Quantitative Amplification Procedures
			29.3.9 In Situ PCR
			29.3.10 Other Approaches
		29.4 Contamination Problems
			29.4.1 Avoiding Contamination
			29.4.2 Decontamination
		29.5 Applications
			29.5.1 Detection of Infectious Diseases
			29.5.2 Detection of Genetic Defects
			29.5.3 The Human Genome Project
		29.6 Alternative Amplification Procedures
			29.6.1 Nucleic Acid Sequence-Based Amplification (NASBA)
			29.6.2 Strand Displacement Amplification (SDA)
			29.6.3 Helicase-Dependent Amplification (HDA)
			29.6.4 Ligase Chain Reaction (LCR)
			29.6.5 Qβ Amplification
			29.6.6 Branched DNA Amplification (bDNA)
		29.7 Prospects
		Further Reading
	Chapter 30: DNA Sequencing
		30.1 Gel-Supported DNA Sequencing Methods
			30.1.1 Sequencing according to Sanger: The Dideoxy Method
			30.1.2 Labeling Techniques and Methods of Verification
			30.1.3 Chemical Cleavage according to Maxam and Gilbert
		30.2 Gel-Free DNA Sequencing Methods - The Next Generation
			30.2.1 Sequencing by Synthesis
			30.2.2 Single Molecule Sequencing
		Further Reading
	Chapter 31: Analysis of Epigenetic Modifications
		31.1 Overview of the Methods to Detect DNA-Modifications
		31.2 Methylation Analysis with the Bisulfite Method
			31.2.1 Amplification and Sequencing of Bisulfite-Treated DNA
			31.2.2 Restriction Analysis after Bisulfite PCR
			31.2.3 Methylation Specific PCR
		31.3 DNA Analysis with Methylation Specific Restriction Enzymes
		31.4 Methylation Analysis by Methylcytosine-Binding Proteins
		31.5 Methylation Analysis by Methylcytosine-Specific Antibodies
		31.6 Methylation Analysis by DNA Hydrolysis and Nearest Neighbor-Assays
		31.7 Analysis of Epigenetic Modifications of Chromatin
		31.8 Chromosome Interaction Analyses
		31.9 Outlook
		Further Reading
	Chapter 32: Protein-Nucleic Acid Interactions
		32.1 DNA-Protein Interactions
			32.1.1 Basic Features for DNA-Protein Recognition: Double-Helical Structures
			32.1.2 DNA Curvature
			32.1.3 DNA Topology
		32.2 DNA-Binding Motifs
		32.3 Special Analytical Methods
			32.3.1 Filter Binding
			32.3.2 Gel Electrophoresis
			32.3.3 Determination of Dissociation Constants
			32.3.4 Analysis of DNA-Protein Complex Dynamics
		32.4 DNA Footprint Analysis
			32.4.1 DNA Labeling
			32.4.2 Primer Extension Reaction for DNA Analysis
			32.4.3 Hydrolysis Methods
			32.4.4 Chemical Reagents for the Modification of DNA-Protein Complexes
			32.4.5 Interference Conditions
			32.4.6 Chemical Nucleases
			32.4.7 Genome-Wide DNA-Protein Interactions
		32.5 Physical Analysis Methods
			32.5.1 Fluorescence Methods
			32.5.2 Fluorophores and Labeling Procedures
			32.5.3 Fluorescence Resonance Energy Transfer (FRET)
			32.5.4 Molecular Beacons
			32.5.5 Surface Plasmon Resonance (SPR)
			32.5.6 Scanning Force Microscopy (SFM)
			32.5.7 Optical Tweezers
			32.5.8 Fluorescence Correlation Spectroscopy (FCS)
		32.6 RNA-Protein Interactions
			32.6.1 Functional Diversity of RNA
			32.6.2 RNA Secondary Structure Parameters and unusual Base Pairs
			32.6.3 Dynamics of RNA-Protein Interactions
		32.7 Characteristic RNA-Binding Motifs
		32.8 Special Methods for the Analysis of RNA-Protein Complexes
			32.8.1 Limited Enzymatic Hydrolyses
			32.8.2 Labeling Methods
			32.8.3 Primer Extension Analysis of RNA
			32.8.4 Customary RNases
			32.8.5 Chemcal Modification of RNA-Protein Complexes
			32.8.6 Chemical Crosslinking
			32.8.7 Incorporation of Photoreactive Nucleotides
			32.8.8 Genome-Wide Identification of Transcription Start Sites (TSS)
		32.9 Genetic Methods
			32.9.1 Tri-hybrid Method
			32.9.2 Aptamers and the Selex Procedure
			32.9.3 Directed Mutations within Binding Domains
		Further Reading
Part V: Functional and Systems Analytics
	Chapter 33: Sequence Data Analysis
		33.1 Sequence Analysis and Bioinformatics
		33.2 Sequence: An Abstraction for Biomolecules
		33.3 Internet Databases and Services
			33.3.1 Sequence Retrieval from Public Databases
			33.3.2 Data Contents and File Format
			33.3.3 Nucleotide Sequence Management in the Laboratory
		33.4 Sequence Analysis on the Web
			33.4.1 EMBOSS
		33.5 Sequence Composition
		33.6 Sequence Patterns
			33.6.1 Transcription Factor Binding Sites
			33.6.2 Identification of Coding Regions
			33.6.3 Protein Localization
		33.7 Homology
			33.7.1 Identity, Similarity, Homology
			33.7.2 Optimal Sequence Alignment
			33.7.3 Alignment for Fast Database Searches: BLAST
			33.7.4 Profile-Based Sensitive Database Search: PSI-BLAST
			33.7.5 Homology Threshold
		33.8 Multiple Alignment and Consensus Sequences
		33.9 Structure Prediction
		33.10 Outlook
	Chapter 34: Analysis of Promoter Strength and Nascent RNA Synthesis
		34.1 Methods for the Analysis of RNA Transcripts
			34.1.1 Overview
			34.1.2 Nuclease S1 Analysis of RNA
			34.1.3 Ribonuclease-Protection Assay (RPA)
			34.1.4 Primer Extension Assay
			34.1.5 Northern Blot and Dot- and Slot-Blot
			34.1.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR and RT-qPCR)
		34.2 Analysis of RNA Synthesis In Vivo
			34.2.1 Nuclear-run-on Assay
			34.2.2 Labeling of Nascent RNA with 5-Fluoro-uridine (FUrd)
		34.3 In Vitro Transcription in Cell-Free Extracts
			34.3.1 Components of an In Vitro Transcription Assay
			34.3.2 Generation of Transcription-Competent Cell Extracts and Protein Fractions
			34.3.3 Template DNA and Detection of In Vitro Transcripts
		34.4 In Vivo Analysis of Promoter Activity in Mammalian Cells
			34.4.1 Vectors for Analysis of Gene-Regulatory cis-Elements
			34.4.2 Transfer of DNA into Mammalian Cells
			34.4.3 Analysis of Reporter Gene Expression
		Further Reading
	Chapter 35: Fluorescent In Situ Hybridization in Molecular Cytogenetics
		35.1 Methods of Fluorescent DNA Hybridization
			35.1.1 Labeling Strategy
			35.1.2 DNA Probes
			35.1.3 Labeling of DNA Probes
			35.1.4 In Situ Hybridization
			35.1.5 Evaluation of Fluorescent Hybridization Signals
		35.2 Application: FISH and CGH
			35.2.1 FISH Analysis of Genomic DNA
			35.2.2 Comparative Genomic Hybridization (CGH)
		Further Reading
	Chapter 36: Physical and Genetic Mapping of Genomes
		36.1 Genetic Mapping: Localization of Genetic Markers within the Genome
			36.1.1 Recombination
			36.1.2 Genetic Markers
			36.1.3 Linkage Analysis - the Generation of Genetic Maps
			36.1.4 Genetic Map of the Human Genome
			36.1.5 Genetic Mapping of Disease Genes
		36.2 Physical Mapping
			36.2.1 Restriction Mapping of Whole Genomes
			36.2.2 Mapping of Recombinant Clones
			36.2.3 Generation of a Physical Map
			36.2.4 Identification and Isolation of Genes
			36.2.5 Transcription Maps of the Human Genome
			36.2.6 Genes and Hereditary Disease - Search for Mutations
		36.3 Integration of Genome Maps
		36.4 The Human Genome
		Further Reading
	Chapter 37: DNA-Microarray Technology
		37.1 RNA Analyses
			37.1.1 Transcriptome Analysis
			37.1.2 RNA Splicing
			37.1.3 RNA Structure and Functionality
		37.2 DNA Analyses
			37.2.1 Genotyping
			37.2.2 Methylation Studies
			37.2.3 DNA Sequencing
			37.2.4 Comparative Genomic Hybridization (CGH)
			37.2.5 Protein-DNA Interactions
		37.3 Molecule Synthesis
			37.3.1 DNA Synthesis
			37.3.2 RNA Production
			37.3.3 On-Chip Protein Expression
		37.4 Other Approaches
			37.4.1 Barcode Identification
			37.4.2 A Universal Microarray Platform
		37.5 New Avenues
			37.5.1 Structural Analyses
			37.5.2 Beyond Nucleic Acids
		Further Reading
	Chapter 38: The Use of Oligonucleotides as Tools in Cell Biology
		38.1 Antisense Oligonucleotides
			38.1.1 Mechanisms of Antisense Oligonucleotides
			38.1.2 Triplex-Forming Oligonucleotides
			38.1.3 Modifications of Oligonucleotides to Decrease their Susceptibility to Nucleases
			38.1.4 Use of Antisense Oligonucleotides in Cell Culture and in Animal Models
			38.1.5 Antisense Oligonucleotides as Therapeutics
		38.2 Ribozymes
			38.2.1 Discovery and Classification of Ribozymes
			38.2.2 Use of Ribozymes
		38.3 RNA Interference and MicroRNAs
			38.3.1 Basics of RNA Interference
			38.3.2 RNA Interference Mediated by Expression Vectors
			38.3.3 Uses of RNA Interference
			38.3.4 microRNAs
		38.4 Aptamers: High-Affinity RNA- and DNA-Oligonucleotides
			38.4.1 Selection of Aptamers
			38.4.2 Uses of Aptamers
		38.5 Genome Editing with CRISPR/Cas9
		38.6 Outlook
		Further Reading
	Chapter 39: Proteome Analysis
		39.1 General Aspects in Proteome Analysis
		39.2 Definition of Starting Conditions and Project Planning
		39.3 Sample Preparation for Proteome Analysis
		39.4 Protein Based Quantitative Proteome Analysis (Top-Down Proteomics)
			39.4.1 Two-Dimensional-Gel-Based Proteomics
			39.4.2 Two-Dimensional Differential Gel Electrophoresis (2D DIGE)
			39.4.3 Top-Down Proteomics using Isotope Labels
			39.4.4 Top-Down Proteomics using Intact Protein Mass Spectrometry
			39.4.5 Concepts in Intact Protein Mass Spectrometry
		39.5 Peptide Based Quantitative Proteome Analysis (Bottom-Up Proteomics)
			39.5.1 Introduction
			39.5.2 Bottom-Up Proteomics
			39.5.3 Complexity of the Proteome
			39.5.4 Bottom-Up Proteomic Strategies
			39.5.5 Peptide Quantification
			39.5.6 Data Dependent Analysis (DDA)
			39.5.7 Selected Reaction Monitoring
			39.5.8 SWATH-MS
			39.5.9 Summary
			39.5.10 Extensions
		39.6 Stable Isotope Labeling in Quantitative Proteomics
			39.6.1 Stable Isotope Label in Top-Down Proteomics
			39.6.2 Stable Isotope Labeling in Bottom-Up Proteomics
		Further Reading
	Chapter 40: Metabolomics and Peptidomics
		40.1 Systems Biology and Metabolomics
		40.2 Technological Platforms for Metabolomics
		40.3 Metabolomic Profiling
		40.4 Peptidomics
		40.5 Metabolomics - Knowledge Mining
		40.6 Data Mining
		40.7 Fields of Application
		40.8 Outlook
		Further Reading
	Chapter 41: Interactomics - Systematic Protein-Protein Interactions
		41.1 Protein Microarrays
			41.1.1 Sensitivity Increase through Miniaturization - Ambient Analyte Assay
			41.1.2 From DNA to Protein Microarrays
			41.1.3 Application of Protein Microarrays
		Further Reading
	Chapter 42: Chemical Biology
		42.1 Chemical Biology - Innovative Chemical Approaches to Study Biological Phenomena
		42.2 Chemical Genetics - Small Organic Molecules for the Modulation of Protein Function
			42.2.1 Study of Protein Functions with Small Organic Molecules
			42.2.2 Forward and Reverse Chemical Genetics
			42.2.3 The Bump-and-Hole Approach of Chemical Genetics
			42.2.4 Identification of Kinase Substrates with ASKA Technology
			42.2.5 Switching Biological Systems on and off with Small Organic Molecules
		42.3 Expressed Protein Ligation - Symbiosis of Chemistry and Biology for the Study of Protein Functions
			42.3.1 Analysis of Lipid-Modified Proteins
			42.3.2 Analysis of Phosphorylated Proteins
			42.3.3 Conditional Protein Splicing
		Further Reading
	Chapter 43: Toponome Analysis
		``Life is Spatial´´
		43.1 Antibody Based Toponome Analysis using Imaging Cycler Microscopy (ICM)
			43.1.1 Concept of the Protein Toponome
			43.1.2 Imaging Cycler Robots: Fundament of a Toponome Reading Technology
			43.1.3 Summary and Outlook
		Acknowledgements
		43.2 Mass Spectrometry Imaging
			43.2.1 Analytical Microprobes
			43.2.2 Mass Spectrometric Pixel Images
			43.2.3 Achievable Spatial Resolution
			43.2.4 SIMS, ME-SIMS, and Cluster SIMS Imaging: Enhancing the Mass Range
			43.2.5 Lateral Resolution and Analytical Limit of Detection
			43.2.6 Coarse Screening by MS Imaging
			43.2.7 Accurate MALDI Mass Spectrometry Imaging
			43.2.8 Identification and Characterization of Analytes
		Further Reading
Appendix 1: Amino Acids and Posttranslational Modifications
Appendix 2: Symbols and Abbreviations
Appendix 3: Standard Amino Acids (three and one letter code)
Appendix 4: Nucleic Acid Bases
Index
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