Fluorescent Proteins: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Using FPbase: The Fluorescent Protein Database
1 Introduction
2 Materials
3 Methods
3.1 Finding a Fluorescent Protein in the Database
3.1.1 Basic Search Field
3.1.2 Advanced Search
3.1.3 FP Properties Table
3.1.4 Interactive FP Properties Chart
3.1.5 Browsing by Lineage or Mutation
3.1.6 FP BLAST
3.1.7 Protein Comparison Tool
3.2 Creating, Sharing, and Downloading Custom FP Collections
3.3 Using the Spectra Viewer
3.3.1 Adding Spectra to the Viewer
3.3.2 Customizing Display and Interacting with the Viewer
3.3.3 Evaluating Excitation and Collection Efficiency
3.3.4 Downloading, Exporting, and Sharing Spectra
3.4 FPbase Microscopes
3.4.1 Gather Information About Your Optical Components
3.4.2 Entering Microscope Information at FPbase
3.4.3 Efficiency Calculations
3.4.4 Efficiency Reports
3.4.5 Allowing Others to Manage Your Microscope
3.4.6 Using Microscope Pages in Publications
3.5 Using the FRET Calculator
4 Notes
4.1 Glossary and Database Schema
4.1.1 Protein Attributes
4.1.2 Fluorescence Attributes
4.1.3 OSER Measurements
4.1.4 Lineages
4.2 Caveats on Interpreting Photostability Measurements
4.3 Caveats on Interpreting Brightness and Efficiency Calculations
4.4 Submitting Data to the Database
4.4.1 Correcting Errors
4.4.2 Submitting New Proteins
4.4.3 Submitting Spectra
References
Chapter 2: Selecting the Fluorescent Protein for In Vivo Imaging Experiments
1 Introduction
2 FP Characteristics to Be Considered
2.1 Fluorescent Protein Expression
2.2 Brightness and Quantum Yield
2.3 Photostability
2.4 Phototoxicity
2.5 pH Sensitivity
2.6 Oligomerization
2.7 Maturation Time
2.8 Selecting Proteins for Multicolor Imaging
References
Chapter 3: Advanced Fluorescence Microscopy Methods to Study Dynamics of Fluorescent Proteins In Vivo
1 Fluorescent Lifetime and Hyperspectral Imaging for Fluorescent Protein Quantitative Imaging
2 FLIM-Phasor for FRET Experiments on Biosensors and Fluorescent Proteins
3 Hyperspectral Imaging and the Spectral Phasor Approach for Fluorescent Protein Dynamics
4 Fluorescence Correlation Spectroscopy (FCS) Methods
5 Raster Scanning Image Correlation Spectroscopy (RICS) Method
References
4: Directed Evolution of Fluorescent Proteins in Bacteria
1 Introduction
2 Materials
2.1 Construction of the DNA Library Encoding Fluorescent Proteins
2.2 Electroporation of the DNA Library into E. coli
2.3 Purification of the Fluorescent Protein
2.4 Characterizing the Biophysical Properties of Evolved Fluorescent Proteins
3 Methods
3.1 Construction of the DNA Library Encoding Fluorescent Proteins
3.1.1 Error-Prone PCR, DNA Extraction, and Gel Purification
3.1.2 Placing the DNA Library Encoding Fluorescent Proteins into the DNA Plasmid
3.1.3 Electroporation of the DNA Library into E. coli
3.2 Fluorescent Screening of Bacteria Expressing Fluorescent Proteins
3.2.1 First Fluorescent Screen for Blueshifted Fluorescent Proteins
3.2.2 Second Fluorescent Screen for Blueshifted Fluorescent Proteins
3.3 DNA Sequence Analysis to Determine Amino Acid Mutations
3.4 Fluorescence Screening of Fluorescent Proteins from Multiple Rounds of Directed Evolution
3.5 Purification of the Fluorescent Protein
3.5.1 Plasmid DNA Transformation of E. coli and Growth
3.5.2 Bacteria Cell Lysis to Free Fluorescent Protein
3.5.3 Nickel-NTA Agarose Purification of His-Tagged Fluorescent Protein
3.5.4 Buffer Exchange
3.6 Biophysical Characterization of the Evolved Fluorescent Proteins
3.6.1 Fluorescent Protein and Standard Solution Absorbance Matching and Extinction Coefficient Determination
3.6.2 Fluorescent Protein and Standard Solution Fluorescence Measurements to Determine the Quantum Yield
4 Notes
References
Chapter 5: Fluorescent Proteins: Crystallization, Structural Determination, and Nonnatural Amino Acid Incorporation
1 Introduction
2 Materials
2.1 E. coli Transformation and Protein Expression
2.2 Protein Extraction and Purification
2.3 Protein Analysis
2.4 Crystallography
3 Methods
3.1 Transformation and Protein Expression
3.1.1 Transformation
3.1.2 Protein Expression
3.2 Protein Extraction and Purification
3.2.1 Cell Lysis by French Press
3.2.2 Immobilized Metal Affinity Chromatography (IMAC)
3.2.3 Size-Exclusion Chromatography (SEC)
3.3 Spectral Analysis of Purified FPs
3.3.1 UV-Visible Absorption Spectroscopy and Calculation of Extinction Coefficients
3.3.2 Fluorescence Spectroscopy
3.4 Crystallography
3.4.1 Crystal Formation and Harvesting
3.4.2 Data Acquisition and Refinement
3.4.3 Inserting Nonstandard Residues and Ligands
4 Notes
References
Chapter 6: Development and Characterization of Flavin-Binding Fluorescent Proteins, Part I: Basic Characterization
1 Introduction
2 Materials
2.1 Cloning
2.2 Bacterial Expression
2.3 Cell Disruption
2.4 Protein Purification
2.5 Measurement of Absorption Spectra and Chromophore Load
2.6 Measurement of Fluorescence Spectra
2.7 Measurement of Thermal Stability
2.8 Fluorescence Microscopy
3 Methods
3.1 Identification of a LOV Domain Sequence
3.2 Gene Cloning
3.3 Bacterial Expression
3.4 Protein Purification
3.5 Measurement of Absorption Spectra
3.6 Measurement of Fluorescence Spectra
3.7 Determination of Chromophore Load from Absorption Spectra
3.8 Measurement of Thermal Stability
3.9 Fluorescence Microscopy
3.9.1 Transfection and Preparation for Microscopy of Mammalian Cells
3.9.2 Transformation and Preparation for Microscopy of E. coli Cells
3.9.3 Imaging of FbFP Inside the Cells
4 Notes
References
Chapter 7: Development and Characterization of Flavin-Binding Fluorescent Proteins, Part II: Advanced Characterization
1 Introduction
2 Materials
2.1 Determination of Flavin Species Distribution by High-Performance Liquid Chromatography
2.2 Determination of FbFP Fluorescence Brightness
2.2.1 Determination of Molar Extinction Coefficient
2.2.2 Determination of Fluorescence Quantum Yield
2.3 Determination of In Vitro Photostability
2.4 Determination of Fluorescence Lifetime
2.5 Quantification of In Vitro Singlet Oxygen (1O2) Production
2.6 Quantification of In Vitro Hydrogen Peroxide (H2O2) Production
2.7 Cultivation of Bacteria and Photosensitizer Expression
2.8 Qualitative and Quantitative Determination of Photosensitizer Phototoxicity
2.9 Fluorescence Imaging of Cell Killing by a Photosensitizer
3 Methods
3.1 Determination of Flavin Species Distribution by HPLC
3.2 Determination of Fluorescence Brightness
3.2.1 Determination of Extinction Coefficient
3.2.2 Determination of Fluorescence Quantum Yield
3.3 Measurement of In Vitro Photostability
3.4 Determination of Fluorescence Lifetime
3.5 Quantification of In Vitro 1O2 Production
3.6 Quantification of PS-Catalyzed Hydrogen Peroxide (H2O2) Production
3.7 Expression of Photosensitizers for Measurements of Phototoxicity
3.7.1 Cultivation of E. coli for Measurement of Intracellular Phototoxicity
3.7.2 Cultivation for Measurement of Extracellular Phototoxicity
3.8 Qualitative Determination of Intra- and Extracellular PS Phototoxicity
3.8.1 Plate Spot Assays for Comparing Intracellular Phototoxicities
3.8.2 Plate Spot Assays for Comparing Extracellular Phototoxicities
3.8.3 Propidium Iodide Assays for Detecting Phototoxicities at Single Cell Level
3.9 Quantitative Determination of Intra- and Extracellular PS Phototoxicity
3.9.1 Determination of the Cell Viability Rate (Colony Forming Units) for Evaluating Intracellular Phototoxicity
3.9.2 Spectrometric Analysis of Light-Induced Cell Death by Intracellular PS Using Propidium Iodide Assay
3.9.3 Flow-Cytometric Analysis of Light-Induced Cell Death by Intracellular PS Using Propidium Iodide Assay
3.9.4 Spectrometric Analysis of Light-Induced Cell Death by Extracellular PS Using Propidium Iodide Assay
3.10 Fluorescence Imaging of Mammalian Cell Killing by PS FbFPs
3.10.1 Cell Transfection
3.10.2 Cell Preparation for Photodynamic Action and Fluorescence Microscopy
3.10.3 Irradiation for and Imaging of Photodynamic Action
3.11 Fluorescence Imaging of Bacteria Killing by PS FbFPs
4 Notes
References
Chapter 8: CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line
1 Introduction
2 Materials
2.1 sgRNA Preparation
2.2 Stable Cell Line Creation
2.3 Cell Cycle Synchronization
2.4 RNP Complex Assembly
2.5 Nucleofection
2.6 Flow Cytometry
2.7 Fluorescence Microscopy
3 Methods
3.1 Pre-experiment Reagent and Cell Preparation
3.1.1 HDR Repair Template Design
3.1.2 sgRNA Preparation
3.1.3 Stable Cell Line Generation
3.1.4 Cell Cycle Synchronization
3.2 Knock-in Experiment
3.2.1 RNP Complex Assembly
3.2.2 Nucleofection
3.3 Post-genome Editing Analysis
3.3.1 Flow Cytometry
3.3.2 Fluorescence Microscopy
4 Notes
References
Chapter 9: Fluorescent Protein-Based Approaches for Subcellular Protein Localization in Plants
1 Introduction
2 Materials
2.1 Particle Bombardment
2.2 Protoplast Isolation and Transformation
2.3 Agrobacterium Infiltration
3 Methods
3.1 Fluorescent Protein Fusion and Construct Preparation
3.2 Particle Bombardment
3.3 Protoplast Isolation
3.4 Protoplast Transformation
3.5 Agrobacterium-Mediated Transient Transformation
4 Notes
References
Chapter 10: Growth Rate Evaluation of the Budding Yeast Saccharomyces cerevisiae Cells Carrying Endogenously Expressed Fluores...
1 Introduction
2 Materials
2.1 Yeast Strains and Media
2.2 Equipment
2.3 Software
3 Methods
3.1 Preparing Yeast for the Growth Assay
3.2 Preparing a 24-Well Plate for Assay
3.3 Growth Assay with Gen5
3.4 Analysis
4 Notes
References
Chapter 11: DNA Visualization Using Fluorescent Proteins
1 Introduction
2 Materials
2.1 DNA Binding Peptides/Proteins
2.1.1 Primers
2.1.2 Cloning Materials
2.1.3 Protein Expression and Purification
2.2 Microfluidic Device and Microscopy
3 Methods
3.1 FP-DBP Cloning and Expression
3.1.1 PCR for the Synthesis of Genes of DNA Binding Fluorescent Proteins
3.1.2 An Alternative Method for Synthesis of Genes of DNA Binding Fluorescent Proteins
3.1.3 Transformation of Ligation Products into Competent Cell DH5α
3.1.4 Colony PCR and Sequencing for Confirmation of Insert Genes in pET-15b
3.1.5 The Transformation of Extracted Plasmids into E. coli BL21 (DE3) Strains
3.1.6 Expression of DNA Binding Proteins with Transformed BL21(DE3) Strains
3.1.7 Extraction and Purification of the FP-DBP
3.1.8 SDS-PAGE for Confirmation of FP-DBP
3.1.9 Storage of DNA Binding Fluorescent Proteins
3.2 Positively Charged Glass Surface Preparation
3.3 Neutravidin-Coated Surface and Flow Chamber Preparation
3.3.1 PEGylated Surface
3.3.2 Protein-Coated Surface (Alternative Way to Prepare Flow Chambers)
3.4 Reversible Staining of DNA with FP-DBP
3.5 Sequence-Specific Staining
3.6 Flow Cell with Surface Immobilization
3.7 Two-Color Staining
3.7.1 Optimization of Two-Color Staining with H-NS-mCherry and BRCA1-eGFP
3.7.2 Observation of λ DNA with Two-Color Staining Using H-NS-mCherry and BRCA1-eGFP
3.8 Two-Color Staining in Flow Cells
4 Notes
References
12: Quantification of Intracellular Citrate Concentrations with Genetically Encoded Biosensors
1 Introduction
2 Materials
2.1 Plasmid Preparation
2.2 Mammalian Cell Culture
2.3 Transfection Reagents
2.4 Working Solution
2.5 Fluorescence Measurement and Data Analysis
3 Methods
3.1 Plasmid Preparation
3.1.1 Preparation from Transformed Bacteria in Agar Stab
3.1.2 Preparation from Purified Plasmids
3.2 Cell Culture and Seeding for Transfection
3.3 Transfection
3.4 Imaging and In Situ Titration
3.5 Data Analysis
4 Notes
References
Chapter 13: Quantification of Redox-Sensitive GFP Cysteine Redox State via Gel-Based Read-Out
1 Introduction
2 Materials
2.1 Affinity-Purified Proteins
2.2 Prereduction or Oxidation of Purified roGFP2
2.3 Glutathionylation of roGPF2 Using GSH/GSSG Buffers with a Specific EGSH
2.4 Labelling of Reduced Thiol Groups
2.5 SDS-Polyacrylamide Gel Components and Gel Imaging
3 Methods
3.1 Preparation of Protein Solutions
3.2 Prereduction or Oxidation of Purified roGFP2
3.3 Equilibration of GSH:GSSG Redox Buffer to a Redox Potential of -280.5 mV
3.4 In Vitro Treatment of roGFP2 to Modify Free Cysteines
3.5 Blocking of Free Thiol Groups with NEM or MPEG-Mal
3.6 Gel-Based Size Shift Read-Out of roGFP2 Redox State
3.7 Quantification of Band Intensities by ImageJ
4 Notes
References
Chapter 14: Assessing In Vivo Oxygen Dynamics Using Plant N-Terminal Degrons in Saccharomyces cerevisiae
1 Introduction
2 Materials
2.1 Media, Buffers, Products, and Enzymes
2.2 Equipment
2.3 Kits
2.4 Yeast and Bacterial Strains
3 Methods
3.1 Cloning of the Constructs in Yeast Expression Plasmids
3.2 Yeast Transformation
3.3 Screening of Luciferase Activity
3.4 Hypoxic Treatments
3.5 Real-Time Oxygen Measurements
3.6 Design of a Dual Fluorescent Oxygen Reporter for Yeast
4 Notes
References
Chapter 15: Osmium-Resistant Fluorescent Proteins and In-Resin Correlative Light-Electron Microscopy of Epon-Embedded Mammalia...
1 Introduction
2 Materials
2.1 Cells, Medium, and Reagents
3 Methods
3.1 Expression of Fluorescent Proteins in Cultured Cells
3.2 Fixation, Staining, and Preparation of Epon-Embedded Samples
4 Notes
References
Chapter 16: Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Proteins
1 Introduction
2 Materials
3 Methods
3.1 The Overview of the FSEC-TS Assay
3.2 Preparation of Cell Lysate or Membrane Fraction from E. coli Cells
3.3 Preparation of Cell Lysate or Membrane Fraction from S. cerevisiae Cells
3.4 Solubilization of Membrane Proteins
3.5 FSEC-TS Assay to Determine the Apparent Tm of Membrane Proteins
3.6 Assessing the Effect of Expression Level on Apparent Tm by FSEC-TS
3.7 FSEC-TS Assay to Screen Detergents
3.8 FSEC-TS Assay to Screen for Stabilizing Lipids
3.9 FSEC-TS Assay to Screen Homologs or Mutants
4 Notes
References
Chapter 17: Determining Protein-Protein Interaction with GFP-Trap Beads
1 Introduction
2 Materials
2.1 Plant Material
2.2 Agrobacterium tumefaciens Strain and Solid Media
2.3 Buffers and Liquid Media
2.4 Miscellaneous
3 Methods
3.1 Growing Agrobacterium for Infiltration
3.2 Agrobacterium Infiltration of N. benthamiana Leaves
3.3 Immunoprecipitation with GFP-Trap Beads
3.4 Protein Extraction and Immunoprecipitation from Stable Transgenic Lines
4 Notes
References
Index