Isotope Labeling of Biomolecules – Labeling Methods
- 1st Edition, Volume 565 - November 3, 2015
- Latest edition
- Editor: Zvi Kelman
- Language: English
Isotope Labeling of Biomolecules – Labeling Methods, the latest volume of the Methods in Enzymology series contains comprehensive information on stable isotope labeling methods… Read more
Isotope Labeling of Biomolecules – Labeling Methods, the latest volume of the Methods in Enzymology series contains comprehensive information on stable isotope labeling methods and applications for biomolecules.
- Contains contributions from leading authorities in the field of isotope labeling of biomolecules
- Informs and updates on the latest developments in the field
- Provides comprehensive information on stable isotope labeling methods and applications for biomolecules
Scientists interested in using stable isotope labeling for structural determination (i.e., SANS and NMR) and other applications (i.e., MS and HDX).
- Preface
- Section I: Labeling in Prokarya
- Chapter One: Robust High-Yield Methodologies for 2H and 2H/15N/13C Labeling of Proteins for Structural Investigations Using Neutron Scattering and NMR
- Abstract
- 1 Introduction
- 2 Media Preparation
- 3 Unlabeled Protein Production
- 4 Deuterated Protein Production
- 5 Multiple Labeling of Proteins for NMR
- 6 Comments on the Method
- 7 Typical Deuteration Levels
- Acknowledgments
- Chapter Two: Protein Labeling in Escherichia coli with 2H, 13C, and 15N
- Abstract
- 1 Introduction
- 2 Selection of Induction Method
- 3 Special Considerations When Labeling with Deuterium
- 4 Plasmid and E. coli Strain Selection
- 5 Determination of Isotope Incorporation
- 6 Acclimation of E. coli to Growth in D2O
- 7 Media Preparation
- 8 Protein Expression
- Acknowledgments
- Chapter Three: Escherichia coli Auxotroph Host Strains for Amino Acid-Selective Isotope Labeling of Recombinant Proteins
- Abstract
- 1 Introduction
- 2 E. coli Auxotrophs for Amino Acid-Selective Isotope Labeling
- 3 Methods
- 4 Conclusions
- Acknowledgments
- Chapter Four: 19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions
- Abstract
- 1 Introduction
- 2 Protocol 1: Biosynthetic Amino Acid Type-Specific Incorporation of 19F-Modified Aromatic Amino Acids
- 3 Protocol 2: Site-Specific Incorporation of Fluorinated Amino Acids Using a Recombinantly Expressed Orthogonal Amber tRNA/tRNA Synthetase Pair in E. coli
- 4 General Considerations for 19F-Observe NMR Experiments
- 5 19F-Modified Protein-Observe NMR Experiments
- 6 NMR Experiments with 19F-Containing Ligands
- Acknowledgments
- Chapter Five: Biopolymer Deuteration for Neutron Scattering and Other Isotope-Sensitive Techniques
- Abstract
- 1 Introduction
- 2 Deuterated Biopolyesters
- 3 Deuterated Chitosan
- 4 Deuterated Cellulose
- Acknowledgments
- Chapter Six: Production of Bacterial Cellulose with Controlled Deuterium–Hydrogen Substitution for Neutron Scattering Studies
- Abstract
- 1 Introduction
- 2 The Occurrence and Properties of Cellulose
- 3 Deuteration of Bacterial Cellulose
- 4 Characterization of Deuterated Cellulose
- Acknowledgments
- Chapter Seven: Isotopic Labeling of Proteins in Halobacterium salinarum
- Abstract
- 1 Introduction
- 2 Growth and Maintenance of Halobacterium salinarum
- 3 Purification of Proteins from Halobacterium salinarum
- 4 Summary
- Acknowledgments
- Chapter Eight: Amino Acid Selective Unlabeling in Protein NMR Spectroscopy
- Abstract
- 1 Introduction
- 2 Method Description
- 3 Applications of Selective Unlabeling
- 4 Conclusions
- Acknowledgments
- Chapter One: Robust High-Yield Methodologies for 2H and 2H/15N/13C Labeling of Proteins for Structural Investigations Using Neutron Scattering and NMR
- Section II: Labeling in Eukarya
- Chapter Nine: Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR
- Abstract
- 1 Introduction
- 2 Expression and Isotope Labeling in P. pastoris: Background
- 3 Isotope Labeling of Membrane Proteins in P. pastoris
- 4 Outlook
- Acknowledgments
- Chapter Ten: Development of Approaches for Deuterium Incorporation in Plants
- Abstract
- 1 Introduction
- 2 Challenges of Plant Cultivation in D2O
- 3 Analysis of Deuterium-Labeled Plant Biomass
- 4 Deuterium Labeling of Plants for Metabolic Studies
- 5 Production of Deuterated Plants for Structural Studies
- Acknowledgments
- Chapter Eleven: Isotope Labeling of Proteins in Insect Cells
- Abstract
- 1 Insect Cells as Expression System
- 2 General Considerations for Isotope Labeling in Insect Cells
- 3 Amino Acid Type-Specific Isotope Labeling in Insect Cells
- 4 Uniform Isotope Labeling in Insect Cells
- 5 Applications
- 6 Protocols
- Chapter Twelve: Effective Isotope Labeling of Proteins in a Mammalian Expression System
- Abstract
- 1 Introduction
- 2 Overview of Mammalian Expression
- 3 Protein Expression
- 4 NMR Characterization of Expressed Protein
- 5 Conclusions
- 6 Materials
- Acknowledgments
- Chapter Nine: Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR
- Section III: In Vitro Labeling
- Chapter Thirteen: Escherichia coli Cell-Free Protein Synthesis and Isotope Labeling of Mammalian Proteins
- Abstract
- 1 Introduction
- 2 The E. coli Cell-Free Protein Synthesis Method
- 3 Stable Isotope Labeling of Proteins
- Acknowledgments
- Chapter Fourteen: Rapid Biosynthesis of Stable Isotope-Labeled Peptides from a Reconstituted In Vitro Translation System for Targeted Proteomics
- Abstract
- 1 Introduction
- 2 Equipment, Materials, and Buffers
- 3 Section 1: DNA Template Preparation
- 4 Section 2: Peptide Synthesis with PURE System
- 5 Section 3: Enrichment and Digestion of Synthesized Peptide
- 6 Section 4: Quantification of PURE-Synthesized Peptide
- 7 An Example
- 8 Summary and Discussion
- Acknowledgment
- Chapter Fifteen: Labeling of Membrane Proteins by Cell-Free Expression
- Abstract
- 1 Introduction
- 2 Core Considerations for the Cell-Free Generation of MP Samples
- 3 Specific Challenges of NMR Studies with MPs
- 4 An Emerging Perspective: NMR with NDs
- 5 Labeling of Cell-Free Synthesized MPs with Stable Isotopes
- 6 Reducing Scrambling Problems
- 7 Perdeuteration of Cell-Free Synthesized MPs
- 8 Conclusion
- Acknowledgments
- Chapter Sixteen: Selective Amino Acid Segmental Labeling of Multi-Domain Proteins
- Abstract
- 1 Introduction
- 2 Methods
- 3 Conclusion
- Acknowledgments
- Chapter Seventeen: Labeling Monosaccharides With Stable Isotopes
- Abstract
- 1 Introduction
- 2 Terminology to Describe Different Monosaccharide Isotopomers
- 3 Introducing 13C into Monosaccharides
- 4 Multiple Labeling of Aldoses Via Chain Inversion
- 5 Labeling at the Internal Carbons of Aldoses
- 6 Extension to Biologically Important Aldoses
- 7 Relative Carbonyl Reactivities in Osones—Synthesis of Labeled 2-Ketoses
- 8 Manipulation of Three-Carbon Building Blocks in Enzyme-Mediated Aldol Condensation
- 9 Manipulation of Isotopically Labeled d-Fructose 17 and l-Sorbose 25
- 10 Concluding Remarks
- Chapter Thirteen: Escherichia coli Cell-Free Protein Synthesis and Isotope Labeling of Mammalian Proteins
- Section IV: RNA Labeling
- Chapter Eighteen: Stable Isotope-Labeled RNA Phosphoramidites to Facilitate Dynamics by NMR
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Protocol
- 5 Step 1: Synthesis of 6-13C-Uridine TOM Phosphoramidite
- 6 Step 2: Synthesis of 6-13C-Cytidine TOM Phosphoramidite
- 7 Step 3: Chemical RNA Synthesis
- 8 Step 4: Applications
- 9 Conclusions
- Acknowledgments
- Chapter Nineteen: In Vivo, Large-Scale Preparation of Uniformly 15N- and Site-Specifically 13C-Labeled Homogeneous, Recombinant RNA for NMR Studies
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Protocol
- 5 Step 1: Pilot of the Expression of the Recombinant tRNA-Scaffold Plasmid in Wild-Type K12 E. coli
- 6 Step 2: Double Selection of High-Expressing E. coli Clones
- 7 Step 3: Large-Scale Expression in Labeled SPG Minimal Media
- 8 Step 4: Total Cellular RNA Extraction
- 9 Step 5a: Purification of the Recombinant tRNA-Scaffold Using Anion-Exchange Chromatography
- 10 Step 5b: Purification of the Recombinant tRNA-Scaffold Using Affinity Chromatography
- 11 Step 6: Excision and Purification of the RNA of Interest
- 12 Step 7: NMR Applications
- 13 Conclusion
- Acknowledgments
- Chapter Twenty: Cut and Paste RNA for Nuclear Magnetic Resonance, Paramagnetic Resonance Enhancement, and Electron Paramagnetic Resonance Structural Studies
- Abstract
- 1 Introduction
- 2 Cut and Paste RNA Approach
- 3 Production of Small (< 10 nts) Isotopically Labeled RNAs
- 4 Protocol A: Production of Small Spin-Labeled RNA Fragments
- 5 Protocol B: Production of Unlabeled and Isotopically Labeled RNA Fragments
- 6 Protocol C: Ligation
- 7 Summary and Outlook
- Acknowledgments
- Chapter Eighteen: Stable Isotope-Labeled RNA Phosphoramidites to Facilitate Dynamics by NMR
- Author Index
Praise for the Series: "Should be on the shelves of all libraries in the world as a whole collection."—Chemistry in Industry "The work most often consulted in the lab." —Enzymologia"The Methods in Enzymology series represents the gold-standard."—Neuroscience
- Edition: 1
- Latest edition
- Volume: 565
- Published: November 3, 2015
- Language: English
ZK
Zvi Kelman
Zvi Kelman is the Director of the Biomolecular Labeling Laboratory (BL2), National Institute of Standards and Technology (NIST). He is also an Adjunct Professor in the Department of Cell Biology and Molecular Genetics at the University of Maryland, College Park and affiliated with the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine. Zvi earned a B.Sc. in Agriculture from the Hebrew University in Jerusalem and a M.Sc. in Cell Biology from the Weizmann Institute of Science. After receiving a Ph.D. in Molecular Biology from Cornell University, he was a Helen Hay Whitney Foundation Post-Doctoral Fellow at Johns Hopkins University and Memorial Sloan-Kettering Cancer Center. Upon completion of his postgraduate training, he became a Life Technologies Professor at the Center for Advanced Research in Biotechnology (CARB), University of Maryland Biotechnology Institute (UMBI). Zvi moved to the University of Maryland, College Park in 2010 as a Professor in the Department of Cell Biology and Molecular Genetics. In 2011 he was recruited to NIST to direct the Biomolecular Labeling Laboratory.
Affiliations and expertise
Director, Biomolecular Labeling Laboratory, NIST-IBBR, Rockville, MD, USARead Isotope Labeling of Biomolecules – Labeling Methods on ScienceDirect