Membrane Lipids: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Methods for Forming Giant Unilamellar Fatty Acid Vesicles
	1 Introduction
	2 Materials
		2.1 Lipids
			2.1.1 Micelle-Based (pH-Decrease) Method
			2.1.2 Paper-Based (pH-Increase) Method
		2.2 Chemicals
		2.3 Equipment
			2.3.1 Micelle-Based (pH-Decrease) Method
			2.3.2 Paper-Based (pH-Increase) Method
			2.3.3 Vesicle Concentration
	3 Methods
		3.1 Buffers
		3.2 Making GUVs Via Micelles (pH-Decrease Method)
			3.2.1 Micelles
			3.2.2 Vesicles
		3.3 Making GUVs Via Paper-Based Thin Film Hydration (pH-Increase Method)
		3.4 Processing Vesicles to Remove Unencapsulated Material
			3.4.1 Concentrate Using Centrifugal Filter
			3.4.2 Concentrate Using Density Difference
	4 Notes
	References
Chapter 2: Preparing Ion Channel Switch Membrane-Based Biosensors
	1 Introduction
	2 Materials
	3 Methods
		3.1 Biosensor Preparation
			3.1.1 Formation of Tethered Components onto Electrodes
		3.2 Preparation of Mobile Phase Lipid Bilayer
		3.3 The Direct ICS Biosensor Preparation
		3.4 The Competitive ICS Biosensor Preparation
		3.5 Sample Preparation
			3.5.1 Measurement Procedure
	4 Notes
	References
Chapter 3: Langmuir-Schaefer Deposition to Create an Asymmetrical Lipopolysaccharide Sparsely Tethered Lipid Bilayer
	1 Introduction
	2 Materials
		2.1 Electrode Selection
		2.2 Cartridge Preparation  Kit
		2.3 Langmuir-Blodgett Trough
		2.4 Solutions
	3 Methods
		3.1 Preparing Langmuir-Trough
		3.2 Langmuir-Shaefer Deposition
		3.3 Preparing Cartridges
		3.4 Testing the Bilayer Using AC Impedance Spectroscopy
	4 Notes
	References
Chapter 4: Electrochemical Impedance Spectroscopy as a Convenient Tool to Characterize Tethered Bilayer Membranes
	1 Introduction
	2 Materials
		2.1 ZView
		2.2 Python
		2.3 Other
		2.4 Solutions
		2.5 Chemicals
		2.6 Working Solutions
		2.7 Proteins (Toxins)
		2.8 Equipment
	3 Methods
		3.1 Sensor Preparation
			3.1.1 Surface Cleaning
			3.1.2 Magnetron Sputtering
			3.1.3 Sensor Surface Functionalization with Thiol  SAM
			3.1.4 Assembly and Storage of Sensors
		3.2 Preparation of Multilamellar Lipid Vesicles (MLVs)
		3.3 Vesicle Fusion and Formation of tBLMs
		3.4 Electrochemical Impedance Spectroscopy (EIS) Measurements
			3.4.1 Setting up EIS Measurements
			3.4.2 Testing the Quality of the Anchor  SAMs
			3.4.3 Real-Time Monitoring of the Vesicle Fusion and tBLM Formation
			3.4.4 Post-Vesicle-Fusion Measurements of EIS Spectra of tBLMs
			3.4.5 Monitoring and Detection of the Activity of Pore-Forming Toxins
		3.5 EIS Data Analysis
			3.5.1 EIS Spectra of  SAMs
			3.5.2 Data Analysis of EIS Spectra of tBLMs
			3.5.3 Evaluation of the Natural Defect Densities in Pristine tBLMs
			3.5.4 Evaluation of the Densities of Toxin Pores in tBLMs (Homogeneous Approximation)
			3.5.5 Evaluation of the Densities of Toxin Pores in tBLMs (Heterogeneous Approximation)
			3.5.6 Detection of Defect Clustering in tBLMs
	4 Notes
	References
Chapter 5: Measuring Voltage-Current Characteristics of Tethered Bilayer Lipid Membranes to Determine the Electro-Insertion Pr...
	1 Introduction
	2 Materials
		2.1 Electrode Selection
		2.2 Cartridge Preparation  Kit
		2.3 Solutions
	3 Methods
		3.1 Preparing Cartridges
		3.2 Creating a Tethered Bilayer Lipid Membrane Using Solvent Exchange
		3.3 Testing the Bilayer Using AC Impedance Spectroscopy
		3.4 Measuring the Voltage-Current Characteristics of Tethered Lipid Membranes
			3.4.1 Setting up the Potentiostat
			3.4.2 The Analysis of the V-I Curve
			3.4.3 Voltage Ramps at High Frequency
		3.5 The Voltage-Current Characteristics of Tethered Lipid Membrane with Analytes Incorporated in their Structure
	4 Notes
	References
Chapter 6: Measuring Activation Energies for Ion Transport Using Tethered Bilayer Lipid Membranes (tBLMs)
	1 Introduction
	2 Materials
		2.1 Membrane Formation
		2.2 Temperature Control Setup
		2.3 Testing the Bilayer Using AC Impedance Spectroscopy
	3 Methods
		3.1 Creating a Tethered Bilayer Lipid Membrane (tBLM) Using Solvent Exchange
		3.2 Measuring the Conductance-Temperature Dependency of Tethered Lipid Membranes
		3.3 Calculating the Activation Energy from the Conductance-Temperature Dependency of Tethered Lipid Membranes
		3.4 Conductance-Temperature Measurement Reversibility
	4 Notes
	References
Chapter 7: Determining the Pore Size of Multimeric Peptide Ion Channels Using Cation Conductance Measures of Tethered Bilayer ...
	1 Introduction
	2 Materials
		2.1 tBLM Preparation Kit and Software
		2.2 Solutions
	3 Methods
		3.1 Assembling SDx Flow Cell Cartridges
		3.2 Creating Tethered Bilayer Lipid Membranes
		3.3 Testing of the Lipid Bilayer
		3.4 Incorporation of Peptide
		3.5 Cation Concentration Gradients
		3.6 Data Analysis
		3.7 Data Interpretation for Upper Pore Size Limit
		3.8 Data Interpretation for Cation Selectivity
	4 Notes
	References
Chapter 8: De-Insertion Current Analysis of Pore-Forming Peptides and Proteins Using Gold Electrode-Supported Lipid Bilayer
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Equipments
		2.3 Experimental Setups for Lipid Bilayer Formation and Channel Current Measurements
	3 Methods
		3.1 PEG-Modified Gold Needle Electrodes
		3.2 Measurements of Reconstitution and De-Insertion Currents of Nanopores
		3.3 DiCA (De-Insertion Current Analysis)
			3.3.1 Step Number Analysis
			3.3.2 Pore Conductance Analysis
	4 Notes
	References
Chapter 9: Drug Meets Monolayer: Understanding the Interactions of Sterol Drugs with Models of the Lung Surfactant Monolayer U...
	1 Introduction
	2 Materials: Simulations and Analysis Programs
	3 Methods
		3.1 Composition of the  LSM
		3.2 Simulations of the LSM Without Drugs (Reference System)
			3.2.1 Set Up of Monolayers at the Vacuum-Water Interface
			3.2.2 Energy Minimization and Equilibration of the  LSM
			3.2.3 Production Runs of the  LSM
		3.3 Simulations of the LSM with Increasing Drug Concentrations (LSM-Systems)
			3.3.1 Drug Concentrations
			3.3.2 Setup of LSM-Drug Systems
			3.3.3 Energy Minimization and Equilibration of LSM-Drug Systems
			3.3.4 Production Runs of the Drug-LSM Systems
		3.4 Analysis
			3.4.1 Area Per Lipid (APL)
			3.4.2 Order Parameter
			3.4.3 Lateral Diffusion and MSD of Lipids
			3.4.4 Clustering of Drugs
			3.4.5 Collapse of Monolayer
	4 Notes
	References
Chapter 10: Establishing a Lipid Bilayer for Molecular Dynamics Simulations
	1 Introduction
	2 Modeling of Lipid Bilayer Structure
		2.1 Unix Environment
		2.2 Topology and Force Field
	3 Computational Details
		3.1 Energy Minimization
		3.2 Equilibration
		3.3 Production  MD
		3.4 Data Gathering
	4 Notes
	References
Chapter 11: Initiating Coarse-Grained MD Simulations for Membrane-Bound Proteins
	1 Introduction
	2 Materials
		2.1 Computer
		2.2 Choice of Operating System
		2.3 Software
			2.3.1 Prerequisites
			2.3.2 Coarse-Grained Simulations
				Linux (Ubuntu)
				Homebrew (Mac)
				If git Is Not Installed
					Linux
					Mac
				After git Is Installed
	3 Methods: Coarse-Grained Simulations
		3.1 Simulation Setup
		3.2 Simulation Analysis
	4 Notes
	References
Chapter 12: Small-Angle Neutron Scattering of Liposomes: Sample Preparation to Simple Modeling
	1 Introduction
	2 Materials
		2.1 Extruder and Filters
		2.2 Small-Angle Neutron Scattering Instruments
	3 Methods
		3.1 Using an Extruder to Prepare Vesicles
		3.2 Small-Angle Neutron Scattering Experiment
		3.3 Modelling the  Data
	4 Notes
	References
Chapter 13: Time-Resolved SANS to Measure Monomer Inter-Bilayer Exchange and Intra-Bilayer Translocation
	1 Introduction
	2 Materials
		2.1 Contrast Match Buffer
	3 Methods
		3.1 Preparation of Large Unilamellar Vesicles
		3.2 TR-SANS Experimentation
		3.3 Data Analysis
			3.3.1 SANS Data Reduction
	4 Notes
	References
Chapter 14: Identifying Membrane Lateral Organization by Contrast-Matched Small Angle Neutron Scattering
	1 Introduction
	2 Materials
		2.1 Small Angle Neutron Scattering Conditions
		2.2 Contrast-Matched (CM) Water
	3 Methods
		3.1 Preparation of Unilamellar Vesicles
		3.2 Small Angle Neutron Scattering
		3.3 Data Analysis-The Porod Invariant
	4 Notes
	References
Chapter 15: Using refnx to Model Neutron Reflectometry Data from Phospholipid Bilayers
	1 Introduction
	2 Materials
	3 Methods
		3.1 Solid-Supported Bilayer
		3.2 Starting refnx
		3.3 Setting Up for Fitting
		3.4 Fitting the Data
		3.5 Assessing Fit Via Markov Chain Resampling
		3.6 MCMC Outputs
	4 Notes
	Appendix
	References
Chapter 16: Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) to Probe Interfacial Water in Floating Bilayer Lipid Me...
	1 Introduction
	2 Materials
		2.1 Cleaning the Prism
		2.2 Deposition of Gold Thin Film on Si Prism
		2.3 Deposition of  fBLM
		2.4 SEIRAS Measurement
	3 Methods
		3.1 Deposition of Gold Film Onto the Planar Surface of Si Hemisphere Prism
		3.2 Preparation of Floating Bilayer Lipid Membrane
		3.3 Measurement of SEIRA Spectra
	4 Notes
	References
Chapter 17: Manipulation of Lipid Membranes with Thermal Stimuli
	1 Introduction
	2 Materials
		2.1 IR Laser-Related Materials and Set-Ups
			2.1.1 IR-VIS Set-Up
				Imaging Module
				IR Module
			2.1.2 Optical Fiber-Associated IR Set-Up
		2.2 Solutions
	3 Methods
		3.1 Preparation of Lipid Samples
		3.2 Preparation of an Open Volume Observation (Sample) Chamber
		3.3 Application of Thermal Gradients
			3.3.1 IR-VIS Through-Objective Set-Up
			3.3.2 Optical Fiber-Associated IR Set-Up
		3.4 Characterization of Local Temperature
			3.4.1 Microthermocouple-Based Measurement
			3.4.2 Preparation of a Microthermocouple
			3.4.3 Gel-Based Temperature Measurement
	4 Notes
	References
Chapter 18: Analyzing Morphological Properties of Early-Stage Toxic Amyloid β Oligomers by Atomic Force Microscopy
	1 Introduction
	2 Materials
		2.1 Amyloid β Solution Preparation
		2.2 Amyloid β Sample Preparation for  AFM
		2.3 AFM Image Processing and Statistical Analysis
	3 Methods
		3.1 Amyloid β Aliquots Preparation
		3.2 Amyloid β Solution Preparation for AFM Measurements
		3.3 Amyloid β Sample Preparation for AFM Measurements
		3.4 AFM Image Processing and Statistical Analysis
	4 Notes
	References
Chapter 19: Formation and Nanoscale Characterization of Asymmetric Supported Lipid Bilayers Containing Raft-Like Domains
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 General Materials
		2.3 Equipment
	3 Methods
		3.1 Preparation of MβCD-SM Complexes
			3.1.1 Preparing SM Multilamellar Vesicles (MLVs)
			3.1.2 Preparing MβCD Stock Solution
			3.1.3 Forming the MβCD-SM Complexes
		3.2 DOPC/Chol (3:1 Mole Ratio) SUVs Preparation
		3.3 Formation of DOPC/Chol (3:1)  SLBs
		3.4 Formation of Asymmetric SLBs Through MβCD-Mediated Lipid Exchange
		3.5 AFM-Imaging and Force Spectroscopy Measurements
	4 Notes
	References
Chapter 20: Rapid FLIM Measurement of Membrane Tension Probe Flipper-TR
	1 Introduction
	2 Materials
		2.1 Labelling Procedure
		2.2 TCSPC Instrumentation
		2.3 Hypo-osmotic Shock Experiments
	3 Methods
		3.1 Instrument Set Up
		3.2 Image Acquisition
		3.3 Analysis and Data Processing
		3.4 Analysis
		3.5 RapidFLIM of Flipper-TR for Fast Spatio-Temporal Membrane Tension Measurement
	4 Notes
	References
Chapter 21: Bacterial Dye Release Measures in Response to Antimicrobial Peptides
	1 Introduction
	2 Materials
	3 Methods
		3.1 Bacterial Growth Conditions
		3.2 Membrane Potential Sensitive Dye DiSC3(5)
		3.3 Nucleic Acid Sytox Green Dye
		3.4 Propidium Iodide (PI) and SYTO9 Dye
	4 Notes
	References
Chapter 22: Quantitative Measurements of Membrane Lipid Order in Yeast and Fungi
	1 Introduction
	2 Materials
		2.1 Cell Culture and Staining
		2.2 Imaging and Analysis
	3 Methods
		3.1 Sample Preparation
		3.2 Mounting Cells into Freshly Prepared Agarose Pad
		3.3 Imaging Acquisition
		3.4 Image Analysis and Quantification
	4 Notes
	References
Index