Integrated Methods in Protein Biochemistry: Part B
- 1st Edition, Volume 679 - January 18, 2023
- Imprint: Academic Press
- Editor: Arun K. Shukla
- Language: English
- Hardback ISBN:9 7 8 - 0 - 3 2 3 - 9 9 2 6 4 - 0
- eBook ISBN:9 7 8 - 0 - 3 2 3 - 9 9 2 6 5 - 7
Integrated Methods in Protein Biochemistry: Part B, Volume 678 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting inter… Read more
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Request a sales quoteIntegrated Methods in Protein Biochemistry: Part B, Volume 678 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of topics, including Precise modification of native proteins, purification, and analysis of bioconjugates, NanoBiT-based methods to monitor the activation and modulation of RTKs, The interplay of G-protein βγ subunit and PLC-β enzyme in PIP2 hydrolysis and downstream signaling, Structure and function of bacterial secretion system, Tools and protocols for probing protein sumoylation, Spectroscopic analysis of cysteine dioxygenase: a mammalian thiol-dioxygenase, DeGlyPHER: MS-based analysis of viral spike N-glycoforms, and more.
Additional sections cover Covalent protein painting: MS-based protein footprinting, Characterization of GPCR signaling complexes using negative-staining electron microscopy, Probing protein misfolding and dissociation with free electron laser, Optimized protocol for the characterization of Cas12a activities, Proximity proteomics for the identification and characterization of extracellular vesicles, Structural and Functional characterization of lytic polysaccharide monooxygenases, and much more.
- Provides the authority and expertise of leading contributors from an international board of authors
- Presents the latest release in Methods in Enzymology series
- Includes the latest information on Integrated Methods in Protein Biochemistry
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
- Cover
- Title page
- Table of Contents
- Copyright
- Contributors
- Preface
- Chapter One: Biochemical analysis of protein–protein interfaces underlying the regulation of bacterial secretion systems
- Abstract
- 1: Introduction
- 2: Case study 1. Quantifying homodimeric interactions using FRET-based in vitro titration
- 3: Case study 2. Using in vitro FRET binding experiments to study competing biomolecular interactions
- 4: Case studies 3 to 6: Mapping novel protein-protein-interfaces through site-directed mutagenesis coupled with in vitro assays
- 5: Case study 3. Examining the role of ExsD trimerization in the inhibition of ExsA-dependent transcription
- 6: Case Study 4. Examining the functional role of the extensive coiled coil region in ExsD through domain deletion with a poly-glycine linker
- 7: Case study 5. Using proline mutations to examine intermolecular backbone contacts between ExsD and ExsA
- 8: Case study 6. Using BACTH assay to map protein-protein interfaces
- References
- Chapter Two: Quantitative structural proteomics in living cells by covalent protein painting
- Abstract
- 1: Introduction
- 2: Experiment layout and general considerations
- 3: General equipment and chemical supplies
- 4: Initial labeling step
- 5: Protein extraction
- 6: Endoproteolytic digestion
- 7: Second labeling, sample cleanup
- 8: Preparation for mass spectrometric measurement and bottom-up proteomic analysis of sample
- 9: Quantification of lysine site accessibility and interpretation of results
- 10: Summary and conclusions
- References
- Chapter Three: Probing protein misfolding and dissociation with an infrared free-electron laser
- Abstract
- 1: Introduction
- 2: Preparation of cells having aggregates
- 3: Dissociation of aggregates by FEL
- 4: MD simulation for the dissociation of protein aggregates with an IR-FEL
- 5: Summary and conclusions
- References
- Chapter Four: Optimized protocols for the characterization of Cas12a activities
- Abstract
- 1: Introduction
- 2: Recombinant protein purification
- 3: Preparation of crRNA
- 4: Activity assays to characterize Cas12a properties
- 5: Quantification of Cas12a activity
- 6: Kinetic analysis
- 7: Conclusions
- References
- Chapter Five: Proximity Labeling and Proteomics: Get to Know Neighbors
- Abstract
- 1: Introduction
- 2: Membrane proximity labeling
- 3: Intracellular proximity labeling
- 4: Proteomics for proximity labeling
- 5: Summary and conclusions
- References
- Chapter Six: Looking at LPMO reactions through the lens of the HRP/Amplex Red assay
- Abstract
- 1: Introduction
- 2: The HRP/Amplex Red assay: Basic principles and pitfalls
- 3: Understanding variations in LPMO performance by measuring in situ H2O2 generation
- 4: The effect of free copper ions on LPMO reactions
- 5: Concluding remarks
- 6: Microtiter plate-based protocol for measuring in situ H2O2 generation in LPMO reactions
- References
- Chapter Seven: Bioinformatic prediction and experimental validation of RiPP recognition elements
- Abstract
- 1: Introduction
- 2: Generation of custom models for RRE prediction and annotation
- 3: Cloning, expression, and purification of RRE domains and precursor peptides
- 4: Fluorescence polarization-binding assays
- 5: Enzyme activity assays and mass spectrometric analysis
- 6: Summary
- References
- Chapter Eight: The preparation of recombinant arginyltransferase 1 (ATE1) for biophysical characterization
- Abstract
- 1: Introduction
- 2: General methods and analysis
- 3: Construct design and protein expression
- 4: Purification of (His)6-tagged ATE1
- 5: Cleavage of (His)6-tagged ATE1
- 6: Size-exclusion purification of ATE1
- 7: Summary and conclusions
- Acknowledgments
- References
- Chapter Nine: Testing anti-cancer drugs with holographic incoherent-light-source quantitative phase imaging
- Abstract
- 1: Introduction
- 2: Protocol
- 3: Summary and conclusions
- Acknowledgments
- References
- Chapter Ten: A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis
- Abstract
- 1: Introduction
- 2: Expression and purification of PheRS protein
- 3: Expression and purification of Mtb tRNAPhe
- 4: PPi production assay (kinetic assay)
- 5: ATP consumption assay (endpoint assay)
- 6: Alternative assay
- 7: Summary
- Acknowledgments
- References
- Chapter Eleven: Site-specific quantitative cysteine profiling with data-independent acquisition-based mass spectrometry
- Abstract
- 1: Introduction
- 2: Quantitative cysteine profiling by DIA-ABPP
- 3: Protocol
- 4: Summary and discussion
- Acknowledgments
- References
- Chapter Twelve: Generation of nucleotide-linked resins for identification of novel binding proteins
- Abstract
- 1: Introduction
- 2: Materials required
- 3: Equipment
- 4: Resin coupling procedure
- 5: Pull-down procedure
- 6: Notes
- References
- Chapter Thirteen: Functional assay of light-induced ion-transport by rhodopsins
- Abstract
- 1: Introduction
- 2: Whole-cell voltage clamp for recording of microbial rhodopsins expressed in mammalian cultured cells
- 3: Ion transport assay of microbial rhodopsins expressed in microbial cells
- Acknowledgment
- References
- Chapter Fourteen: Assay design for analysis of human uracil DNA glycosylase
- Abstract
- 1: Introduction: Biomedical relevance of uracil DNA glycosylase activity
- 2: UNG2 characteristics and enzyme activity assays
- 3: Future utility for assays in discovering UNG2 inhibitors and assessing novel protein-protein interactions
- Acknowledgments
- References
- Chapter Fifteen: Measurement of kinetic isotope effects on peptide hydroxylation using MALDI-MS
- Abstract
- 1: Introduction
- 2: Equipment, buffers and reagents, procedures
- 3: Measurement of KIEs
- 4: Uncoupling of O2 consumption from CTAD hydroxylation
- 5: Autohydroxylation assay and simultaneous detection of hydroxylated CTAD product
- 6: Conclusion
- Acknowledgments
- References
- Chapter Sixteen: Continuous photometric activity assays for lytic polysaccharide monooxygenase—Critical assessment and practical considerations
- Abstract
- 1: Introduction
- 2: Continuous photometric assays
- 3: Continuous turbidimetric assay
- 4: Summary and concluding remarks
- References
- Edition: 1
- Volume: 679
- Published: January 18, 2023
- Imprint: Academic Press
- No. of pages: 420
- Language: English
- Hardback ISBN: 9780323992640
- eBook ISBN: 9780323992657
AS
Arun K. Shukla
Dr. Arun K. Shukla is a world leader in the field of GPCR biology and he is currently a Professor in the Department of Biological Sciences and Bioengineering at the Institute of Technology, Kanpur in India. Dr. Shukla’s research program is focused on understanding the structure, function and regulation of G protein-coupled receptors with a long-term of designing novel therapeutics with minimized side-effects.
Affiliations and expertise
Indian Institute of Technology, Kanpur, IndiaRead Integrated Methods in Protein Biochemistry: Part B on ScienceDirect