دانلود کتاب Plant Proteostasis: Methods and Protocols (Methods in Molecular Biology, 1450)

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Preface
Contents
Contributors
Part I: Ubiquitin Conjugation and Deconjugation Analysis
Chapter 1: Approaches to Determine Protein Ubiquitination Residue Types
1 Introduction
2 Materials
2.1 Extraction of Plant Proteins
2.2 SDS-PAGE
2.3 Immunoblotting Components
2.4 Immuno-precipitation
3 Methods
3.1 Protein Expression and Extraction
3.2 Immuno-precipitation
3.3 In Vivo Detection of Polyubiquitination
3.4 Detection of Polyubiquitination on Cys Residues
3.5 Detection of Polyubiquitination on Ser/Thr Residues
3.6 Western Blotting
4 Notes
References
Chapter 2: Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings
1 Introduction
2 Materials
2.1 Co-IP of CRLs
2.1.1 Plant Material and Growth
2.1.2 Total Protein Extraction
2.1.3 Immuno-precipitation
2.1.4 SDS-PAGE
2.1.5 Immunoblotting and Detection
2.2 In Vivo Ubiquitination Analysis of CRLs
2.2.1 Plant Material and Growth
2.2.2 Total Protein Extraction
2.2.3 Immuno-precipitation
2.2.4 SDS-PAGE
2.2.5 Immunoblot and Detection
3 Methods
3.1 IP of CRLs
3.1.1 Plant Material
3.1.2 Total Protein Extraction
3.1.3 Immuno-precipitation
3.1.4 SDS-PAGE
3.1.5 Immunoblot and Detection
3.2 In Vivo Ubiquitination Analysis of CRLs
3.2.1 Plant Material and Growth
3.2.2 Total Protein Extraction
3.2.3 Immuno-precipitation
3.2.4 SDS-PAGE
3.2.5 Immunoblot and Detection
4 Notes
References
Chapter 3: Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors
1 Introduction
2 Materials
2.1 Saturation and Competition Binding Reactions
2.2 Quantifying Bound Radioligand
2.3 Data Analysis
3 Methods
3.1 Radioactivity Calculations
3.2 Saturation Binding Experiment
3.3 Competition Binding Experiment
3.4 Quantifying Bound Radioligand
3.5 Data Analysis
3.6 Analysis for Saturation Binding
3.7 Analysis for Competition Binding
4 Notes
References
Chapter 4: Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes
1 Introduction
2 Materials
2.1 Recombinant GST-Tagged DUB Purification from Bacteria
2.2 Deubiquitylation Assay (DUB Assay)
2.3 Gradient Gel Electrophoresis
2.4 Protein Transfer and Western Blotting
2.5 CBB Staining
2.6 Fluorescence-­Based DUB Assay
3 Methods
3.1 Recombinant DUB Purification from Bacteria
3.2 Deubiquitylation Assay
3.3 Gradient Gel Electrophoresis
3.4 Protein Transfer and Western Blotting
3.5 CBB Staining
3.6 Fluorescence-Based DUB Assay
4 Notes
References
Chapter 5: Fluorescent Reporters for Ubiquitin-Dependent Proteolysis in Plants
1 Introduction
2 Materials
2.1 PCR and Plasmid Recombination
2.2 Transient Transformation
2.3 SDS-PAGE and Western Blot
3 Methods
3.1 Plasmid Creation
3.2 Transient N. benthamiana Leaf Transformation
3.3 Microscopy Observation
3.4 Western Blot Analysis
4 Notes
References
Chapter 6: Generation of Artificial N-end Rule Substrate Proteins In Vivo and In Vitro
1 Introduction
1.1 Importance of Creating Proteins Comprising Defined N-Termini
1.2 Ubiquitin Fusion Technique
1.3 UFT In Vivo
1.4 UFT In Vitro
1.5 TEV Cleavage
2 Materials
2.1 General Equipment
2.2 Cloning
2.3 Protoplast Isolation (See Table 3)
2.4 PEG-Mediated Transformation of DNA into Protoplasts (See Table 3)
2.5 Analysis of Protein Expression in Protoplasts
2.6 Protein Expression in a Cell-Free System
2.7 Protein Expression in Bacteria
2.8 TEV Cleavage
2.9 Protein Concentration Determination
2.10 SDS-
2.11 Western Transfer of Proteins and Detection
3 Methods
3.1 General Considerations
3.2 Cloning
3.3 Protoplast Isolation
3.4 PEG-Mediated Transformation of DNA into Protoplasts
3.5 Analysis of Protein Expression in Protoplasts via Fluorescence Microscopy
3.6 Analysis of Protein Expression in Protoplasts by Western Blotting
3.7 Protein Expression in a Cell-Free System
3.8 Protein Expression in Bacteria
3.9 TEV Cleavage
3.10 Protein Concentration Determination
3.11 SDS-
3.12 Western Transfer of Proteins and Detection
4 Notes
References
Chapter 7: Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases
1 Introduction
1.1 Peptide Arrays for Protein Interaction Studies
1.2 SPOT Assays on N-End Rule Enzymes
2 Materials
2.1 General Equipment and Infrastructure
2.2 Membrane Activation and Blocking
2.3 Enzyme Binding on SPOT Membranes
2.4 Western Blotting of SPOT-Array-­Bound Proteins
3 Methods
3.1 Membrane Activation and Blocking
3.2 Enzyme Binding on SPOT Membranes
3.3 Western Blotting of SPOT-Array-­Bound Proteins
4 Notes
References
Part II: Ubiquitin-like Protein Conjugation, Deconjugation, and Cell Imaging Studies
Chapter 8: SUMO Chain Formation by Plant Enzymes
1 Introduction
2 Materials
2.1 Components of the SUMOylation System
2.2 Expression of Recombinant Plant SUMOylation Enzymes in E. coli
2.3 Purification of His-Tagged Proteins (See Notes 2 and 3)
2.4 Purification of MBP-Tagged Proteins
2.5 Purification of Untagged SCE1
2.6 FPLC Purification
2.7 In Vitro SUMO Chain Formation
3 Methods
3.1 Expression of Recombinant Plant SUMOylation Enzymes in E. coli
3.2 Purifying His-Tagged Proteins (SUMO and SAE)
3.3 Purification of MBP-Tagged Proteins (PIAL1 and PIAL2)
3.4 Purification of Untagged Proteins (SCE1)
3.5 In Vitro SUMO Chain Formation. Small Scale Reaction for Western Blot Detection (Fig. 1)
3.6 In Vitro SUMO Chain Formation. Large Scale Reaction for Isolation of SUMO Chains
3.7 SUMO Protease Activity Test Using SUMO Chains (Fig. 2)
4 Notes
References
Chapter 9: Kinetic Analysis of Plant SUMO Conjugation Machinery
1 Introduction
2 Materials
2.1 Expression and Purification of Enzymes
2.2 SUMO Conjugation Assays
3 Methods
3.1 Preparation of Recombinant SUMO Machinery Components: Expression and Purification of SUMO Isoforms and the SUMO-­Conjugating Enzyme SCE1
3.2 Preparation of Recombinant SUMO Machinery Components: Expression and Purification of E1-Activating Enzyme Isoforms (SAE2/SAE1a and SAE2/ SAE1b)
3.3 Preparation of Recombinant SUMO Conjugation Substrate: Expression and Purification of GST-CAT3Ct
3.4 In Vitro SUMOylation Assays for Analyzing Distinct SUMO Isoforms
3.5 Quantification of SUMO Conjugation Kinetics
4 Notes
References
Chapter 10: Expression, Purification, and Enzymatic Analysis of Plant SUMO Proteases
1 Introduction
2 Materials
2.1 Bacterial Culture and Purification of Arabidopsis OTS1 SUMO Protease
2.2 SUMO Protease Assay
3 Methods
3.1 Expression of Recombinant SUMO Protease Protein
3.2 Purification of Expressed Protein Using Small Scale Batch Method
3.3 SUMO Protease Activity Assay Using a SUMO Linked Substrate
4 Notes
References
Chapter 11: Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging
1 Introduction
2 Materials
2.1 Vectors
2.2 Plant Tissue
2.3 Bombardment Equipment
2.4 Reagents
2.5 Microscopy Equipment
2.6 Software
3 Methods
3.1 Design and Generation of Fluorescence Chimeric Proteins
3.2 Biolistic Bombardment: Microcarrier Preparation
3.3 Biolistic Bombardment: Coating Washed Microcarriers with DNA
3.4 Performing Bombardment
3.5 Fluorescence Protein Detection and Imaging
3.6 Average Fluorescence Intensity Measurements
3.7 Statistical Analysis of Average Fluorescence Intensity
4 Notes
References
Chapter 12: Biochemical Analysis of Autophagy in Algae and Plants by Monitoring the Electrophoretic Mobility of ATG8
1 Introduction
2 Materials
2.1 Growth Media and Components
2.2 SDS-PAGE Components
2.3 Immunoblot Components
3 Methods
3.1 Preparation of Proteins from Chlamydomonas
3.2 Preparation of Proteins from Arabidopsis
3.3 Separation and Analysis of Proteins by Electrophoresis
3.4 Western Blotting and ATG8 Protein Detection
4 Notes
References
Chapter 13: Detection of Autophagy in Plants by Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Plant Growth Materials
2.2 Seedling Treatment and Staining
2.3 Fluorescence Microscopy
3 Methods
3.1 Plant Materials and Growth Conditions
3.2 Autophagy Activation in Seedlings by Abiotic Stresses (See Note 7)
3.3 Labeling of Autophagosomes in Seedlings by MDC Staining
3.4 Labeling of Autophagosomes in Seedlings with GFP-ATG8 Fusion Protein (See Note 10)
3.5 Visualization of MDC-Stained or GFP-Labeled Autophagosomes by Fluorescence Microscopy
4 Notes
References
Part III: Proteomic Analysis and Other Post-Translational Modification Studies
Chapter 14: Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System
1 Introduction
2 Materials
2.1 Maintenance of Arabidopsis Cell Suspension Culture, Preparation of Arabidopsis Protoplasts and Transfections
2.2 Metabolic Labeling and Pulse-Chase Experiments
2.3 Stability Measurements Using Reporter-­Dependent Assays
3 Methods
3.1 Preparation of Arabidopsis Protoplasts and Transfection
3.2 Pulse-Chase Experiments Using Radioactive Metabolic Labeling of Transfected Protoplasts
3.3 Stability Measurements Using Reporter-­Dependent Assays
4 Notes
References
Chapter 15: Detection and Quantification of Protein Aggregates in Plants
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Protein Extraction and Aggregate Isolation
2.3 Immunoblot
2.4 Silver Stain
3 Methods
3.1 Plant Growth and Senescence Induction
3.2 Cell Fractionation (Scheme of the Process Shown in Fig. 1)
3.3 Aggregate Isolation
3.4 Localization of Particular Proteins in the Aggregate Fraction
3.5 Relative Quantification of Aggregates
4 Notes
References
Chapter 16: Determination of Protein Carbonylation and Proteasome Activity in Seeds
1 Introduction
2 Materials
2.1 Determination of Protein Carbonylation by Spectrophotometry
2.2 Proteasome Activity Profiling
3 Methods
3.1 Determination of Protein Carbonylation by Spectrophotometry
3.1.1 Protein Extraction and Quantification
3.1.2 Determination of Carbonyl Groups
3.2 Proteasome Activity Profiling
3.2.1 Protein Extraction and Quantification
3.2.2 In-gel Proteasome Activity-Based Protein Profiling
4 Notes
References
Chapter 17: Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-Based Protein Profiling in Plants
1 Introduction
2 Materials
2.1 Solutions
2.2 Preparation of Protein Extracts
2.3 Trypsin Digestion and iTRAQ Labeling
2.4 MS/MS Analysis
2.5 Protein Identification
3 Methods
3.1 Sample Preparation
3.2 In-Solution Trypsin Digestion and iTRAQ Labeling
3.3 Nano-HPLC-MS/MS Analysis
3.4 Database Search and Quantitation
3.5 Data Processing and Statistical Analysis
4 Notes
References
Chapter 18: Use of a Phosphatidylinositol Phosphate Affinity Chromatography (PIP Chromatography) for the Isolation of Proteins Involved in Protein Quality Control and Proteostasis Mechanisms in Plants
1 Introduction
2 Materials
2.1 Cell Culture Medium
2.2 Extraction (TNEE) Buffer
2.3 Q and S Sepharose
2.4 IPP Buffer
2.5 Laemmli Sample Buffer
2.6 Bead Washing Buffer
3 Methods
3.1 PIP-Affinity Chromatography
3.2 Cell Culture (See Note 1)
3.3 Cell Extraction
3.4 Sample Fractionation Using Q and S Sepharose
3.5 PIP Incubation
3.5.1 For Small-Scale Trials
3.5.2 For Large-Scale Trials Followed by Mass Spectrometry
4 Notes
References
Chapter 19: In Vivo Radiolabeling of Arabidopsis Chloroplast Proteins and Separation of Thylakoid Membrane Complexes by Blue Native PAGE
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Thylakoid Membrane Isolation
2.3 Sample Preparation
2.4 BN-PAGE (First Dimension)
2.5 Denaturating SDS-PAGE (Second Dimension)
2.6 Silver Staining
2.7 Radiolabeling
2.8 Western Blot Using Semi-dry Electroblotting
3 Methods
3.1 Thylakoid Membrane Isolation of Unlabeled Leaf Material
3.2 Sample Preparation
3.3 BN-PAGE (First Dimension)
3.4 Denaturating SDS-PAGE (Second Dimension)
3.5 Silver Staining
3.6 Radiolabeling
4 Notes
References
Chapter 20: Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Protein Quantification (See Note 5)
2.3 Protein Labeling
2.4 SDS-PAGE and Western Blot
2.5 Antibodies
2.6 Target Detection by Chemiluminescence
2.7 Target Detection by Fluorescence
2.8 Quantification
3 Methods
3.1 Protein Extraction
3.2 Protein Quantification
3.3 Protein Labeling Reactions
3.4 SDS-PAGE and Western Blot
3.5 Antibodies
3.6 Target Detection by Chemiluminescence (See Note 7)
3.7 Target Detection by Fluorescence (See Note 7)
3.8 Quantification
4 Notes
References
Part IV: Bioinformatics Analysis
Chapter 21: Sequence Search and Comparative Genomic Analysis of SUMO-Activating Enzymes Using CoGe
1 Introduction
2 Materials
3 Methods
3.1 Search for SAE1 Sequences in Selected Plant Genomes. The Case of Arabidopsis Thaliana
3.2 Analysis of the Brassicaceae-Specific WGD Leading to the Duplicate Gene Pair SAE1a/SAE1b
3.3 Analysis of the Tandem Duplication Leading to the Gene Duplicate Pair At5g50580–At5g50680 in A. Thaliana Col-0
4 Notes
References
Chapter 22: Studying Evolutionary Dynamics of Gene Families Encoding SUMO-Activating Enzymes with SeaView and ProtTest
1 Introduction
2 Materials
3 Methods
3.1 Sequence Edition and Multiple Alignment with SeaView
3.2 Selection of Best-fit Model of Amino Acid Substitution Using ProtTest
3.3 Phylogenetic Analysis Using PhyML (ML) and NJ
4 Notes
References
Chapter 23: Bioinformatics Tools for Exploring the SUMO Gene Network
1 Introduction
2 Materials
3 Methods
3.1 The SUMO Gene Network
3.2 Managing Datasets
3.2.1 Duplicate Removal
3.2.2 Generate Venn Diagrams
3.3 In Silico Prediction of SUMO Attachment Sites and SIMs
3.4 Comparative Genomics for Ortholog Identification
3.5 Functional Categorization and Gene Network Analysis
4 Notes
References
Index