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|a Arnesen, Thomas.
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|a Protein Termini Part A.
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| 250 |
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|a 1st ed.
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| 264 |
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1 |
|a Chantilly :
|b Elsevier Science & Technology,
|c 2025.
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|c ©2025.
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|a 1 online resource (309 pages).
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|2 onix
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|b Index navigation
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|b Single logical reading order
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|a Methods in Enzymology Series ;
|v v.Volume 718
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| 505 |
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|a Front Cover -- Series Page -- Methods in Enzymology -- Copyright -- Contents -- Contributors -- Chapter One: Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases -- 1 Introduction -- 2 Before you begin -- 2.1 Buffers -- 3 Key resources table -- 4 Materials and equipment -- 4.1 Materials -- 4.2 Equipment -- 4.3 Reagents -- 5 Step-by-step method details -- 5.1 Expression of MetAPs and NATs -- 5.2 Purification of HsMetAP1 and HsMetAP2 -- 6 Expected outcomes -- 6.1 Purification of HsNatA -- 7 Expected outcomes -- 7.1 Purification of HsNatA-HypK -- 8 Expected outcomes -- 8.1 Purification of HsNAA50 -- 9 Expected outcomes -- 10 Optimization and troubleshooting -- 10.1 The expression did not work -- 10.2 Protein is not eluting from the column after protease incubation -- 10.3 MetAP2 shows a degradation pattern on the SDS-gel -- 10.4 The lysate is not moving through the column -- References -- Chapter Two: Purification and activity assays of N-terminal acetyltransferase D -- 1 Introduction -- 2 Preparation of recombinant NatD -- 2.1 Equipment -- 2.2 Material and buffer recipes -- 2.3 Transformation -- 2.4 Expression -- 2.5 Purification -- 2.5.1 Preparation of cell lysate -- 2.5.2 Enrichment of His-TEV-NatD through Ni-NTA column -- 2.5.3 Cleavage of His-TEV tag -- 2.6 Expected outcomes -- 2.7 Notes -- 3 Continuous fluorescence acetyltransferase assay -- 3.1 Equipment -- 3.2 Material and buffer recipes -- 3.3 Fluorescence assay procedures -- 3.3.1 Optimization of ThioGlo4 concentration -- 3.3.2 Standard curve of fluorescence intensity versus [CoASH] -- 3.3.3 Determine the optimal [enzyme] -- 3.3.4 Peptide Km study -- 3.3.5 AcCoA Km study -- 3.3.6 IC50 study -- 3.4 Data analysis -- 3.4.1 AcCoA standard curve -- 3.4.2 Km of peptide or AcCoA -- 3.4.3 IC50 study -- 3.5 Notes -- 4 MALDI-MS acetyltransferase assay.
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| 505 |
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|a 4.1 Equipment -- 4.2 Material and buffer recipes -- 4.3 MALDI-MS assay procedures -- 4.3.1 Peptide Km study -- 4.3.2 IC50 study -- 4.4 Data analysis -- 4.4.1 Peptide Km study -- 4.4.2 IC50 study -- 4.5 Notes -- 5 Summary and conclusions -- Acknowledgment -- References -- Chapter Three: A fluorescent CPM-based in vitro acetylation assay: A tool for assessing N-terminal acetyltransferase activity and profiling compound activity -- 1 Introduction -- 2 Method overview -- 3 Equipment and reagent preparation -- 3.1 Equipment for N-terminal acetylation assay -- 3.1.1 Microplates -- 3.1.2 Plate readers -- 3.2 Preparation of reagents for the N-terminal acetylation assay -- 3.2.1 Expression and purification of N-terminal acetyltransferases A and B -- 3.2.1.1 NatA purification -- 3.2.1.2 NatB purification -- 3.2.1.3 Notes -- 3.2.2 Preparation of reagents for N-terminal acetylation assay -- 3.2.2.1 Ac-CoA -- 3.2.2.2 CoA -- 3.2.2.3 Peptides -- 3.2.2.4 CPM -- 3.2.2.5 Reaction buffer -- 3.2.2.6 Bisubstrate inhibitor (CoA-SASEA) -- 3.2.2.7 Notes -- 4 Readout reaction CPM-CoA -- 4.1 CoA-dependent CPM signal and its temporal stability -- 4.1.1 Procedure -- 4.2 Linear relationship between CoA concentration and CPM fluorescence -- 4.2.1 Procedure -- 4.3 Notes -- 5 Establishing assay conditions and Michaelis-Menten kinetics -- 5.1 Optimization of NatA and NatB concentrations -- 5.1.1 Procedure -- 5.2 Time course with fixed NatA concentration -- 5.2.1 Procedure -- 5.3 Reaction linearity at selected substrate concentrations -- 5.3.1 Procedure -- 5.4 Michaelis-Menten kinetics: Km and kcat for peptide and Ac-CoA -- 5.4.1 Procedure -- 5.5 Notes -- 6 Miniaturization of assay to 384-well format -- 6.1 Procedure -- 6.2 Notes -- 7 Test of NatA inhibition (bisubstrate inhibitor) -- 7.1 Procedure -- 7.2 Notes -- 8 Compound screening -- 8.1 Equipment -- 8.2 Reagents -- 8.3 Procedure.
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| 505 |
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|a 8.4 Notes -- 9 Conclusions and outlook -- Acknowledgments -- References -- Chapter Four: In vitro acetyltransferase activity assays for N-terminal acetyltransferases -- 1 Introduction -- 2 Overview of NAT activity assays -- 3 NAT enzyme preparation -- 4 Activity assays -- 4.1 14C Radioactive acetyltransferase assay -- 4.1.1 Materials and instrumentation -- 4.1.2 Reaction conditions -- 4.2 Fluorescence assay -- 4.2.1 Materials and instrumentation -- 4.2.2 Remove free CoA in AcCoA -- 4.2.3 Reaction conditions -- 4.2.4 Kinetic analysis of Michaelis-Menten constants (Km) -- 4.3 ACSS2 coupled luminescence assay -- 4.3.1 ACSS2 purification -- 4.3.2 Materials and instrumentation -- 4.3.3 Reaction conditions -- 4.4 Comparison between assays -- 5 Prospects and conclusions -- Acknowledgments -- References -- Chapter Five: Parallel reaction monitoring reveals N-terminal acetylation of plastid precursor proteins -- 1 Introduction -- 2 Experimental design -- 3 General considerations -- 4 Protoplast protein import assay -- 4.1 Equipment, materials and buffer recipes -- 4.1.1 Equipment -- 4.1.2 Materials -- 4.1.3 Buffer components -- 4.1.4 Plasmid construction -- 4.2 Procedures -- 4.2.1 Plant growth -- 4.2.2 Protoplast isolation, transformation and protein extraction -- 4.2.3 SDS-PAGE and Immunoblot analysis -- 5 LC-MS/MS and N-terminal peptide identification via Skyline -- 5.1 Equipment, materials, and buffer recipes -- 5.1.1 Equipment -- 5.1.2 Material -- 5.1.3 Buffer components -- 5.2 Procedures -- 5.2.1 Sample preparation and tryptic in-gel digest -- 5.2.2 Targeted LC-MS/MS -- 5.2.3 Data analysis -- 6 Conclusion -- Acknowledgments -- References -- Chapter Six: N-terminal acetylation-specific antibodies: Specificity determination by mass spectrometry and utilization in in vitro acetylation assays -- 1 Introduction -- 2 Methods.
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| 505 |
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|a 3.4.2 SPOT array -- 4 Development of anti-pan-N-fMet-specific antibody using a mixed antigen -- 4.1 Antigen design -- 4.2 Antibody production -- 5 Analysis of protein Nt-formylation -- 5.1 Detection in Escherichia coli -- 5.1.1 Equipment, materials, and buffer recipes -- 5.1.2 Cell growth and lysis -- 5.1.3 Immunoblotting with pan-fMet-specific antibodies -- 5.2 Detection in Salmonella Typhimurium -- 5.2.1 Equipment, materials, and buffer recipes -- 5.2.2 Cell growth -- 5.2.3 Cell lysis -- 5.2.4 Immunoblotting with pan-fMet antibodies -- 5.3 Detection in yeast mitochondria -- 5.3.1 Equipment, materials, and buffer recipes -- 5.3.2 Cell culture -- 5.3.3 Mitochondria fractionation and lysis -- 5.3.4 Immunoblotting of fMet-proteins with pan-fMet-specific antibodies -- 5.4 Detection in human cells -- 5.4.1 Equipment, materials, and buffer recipes -- 5.4.2 Cell culture -- 5.4.3 Cell lysis -- 5.4.4 Immunoblotting with pan-fMet antibodies -- 5.5 Detection of synthetic peptides -- 5.5.1 Equipment, materials, and buffer recipes -- 5.5.2 Peptide conjugation -- 5.5.3 ELISA with pan-fMet-specific antibodies -- 6 Concluding remarks -- Acknowledgments -- Disclosure statement -- References -- Chapter Nine: Chemical proteomic approaches to investigate N-myristoylation -- 1 Introduction -- 2 General proteomic considerations -- 3 Metabolic labelling protocol -- 3.1 Prior considerations -- 3.2 Cell treatment -- 3.2.1 Equipment, buffers and reagents -- 3.2.2 Procedure -- 3.3 Bio-orthogonal click reaction and tag ligation -- 3.3.1 Equipment, buffers and reagents -- 3.3.2 Procedure -- 3.4 Protein enrichment -- 3.4.1 Equipment, buffers and reagents -- 3.4.2 Procedure -- 3.5 Cysteine alkylation and protein digest -- 3.5.1 Equipment, buffers and reagents -- 3.5.2 Procedure -- 3.6 Peptide preparation and LC-MS/MS analysis -- 3.6.1 Equipment, buffers and reagents.
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| 500 |
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|a 3.6.2 Procedure.
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| 520 |
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|a Protein termini represent a major route to protein regulation.From the moment the very first amino acid of a polypeptide chain exits the ribosome there is potential for steering from the cellular environment.
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| 532 |
8 |
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|a Trusted Intermediary contact: t.narup@elsevier.com
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| 532 |
8 |
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|a Publisher contact for further accessibility information: t.narup@elsevier.com
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| 588 |
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|a Description based on publisher supplied metadata and other sources.
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| 650 |
|
0 |
|a Enzymology.
|
| 650 |
|
0 |
|a Proteins
|x Synthesis.
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| 650 |
|
0 |
|a Proteins.
|
| 650 |
|
6 |
|a Enzymologie.
|
| 650 |
|
6 |
|a Protéines
|x Synthèse.
|
| 650 |
|
6 |
|a Protéines.
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| 650 |
|
7 |
|a protein.
|2 aat
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| 655 |
|
7 |
|a Electronic books.
|2 local
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| 700 |
1 |
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|a Thomas Arnesen.
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| 710 |
2 |
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|a ScienceDirect (Online service)
|
| 830 |
|
0 |
|a Methods in Enzymology Series.
|
| 856 |
4 |
0 |
|u http://proxy.library.tamu.edu/login?url=https://www.sciencedirect.com/science/bookseries/00766879/718
|z Connect to the full text of this electronic book
|t 0
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| 880 |
8 |
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|6 505-00/(S
|a 2.1 Peptide dot blot to determine the range of specificity of pan-anti-Nt-Ac antibody -- 2.1.1 Materials and equipment -- 2.1.2 Procedure -- 2.2 Immunoprecipitation with anti-Nt-Ac-Met followed by LC/MS-MS analysis of pulldowns (IP-MS) -- 2.2.1 Materials and equipment -- 2.2.2 Procedure -- 2.2.3 Perseus data analysis -- 2.2.4 Enrichment analysis of Nt-sequence -- 2.3 In vitro peptide Nt-acetylation assay with anti-Nt-Ac-Met as readout -- 2.3.1 Materials and equipment -- 2.3.2 Procedure -- 3 Results and discussion -- 4 Conclusions -- Acknowledgements -- Appendix A. Supporting information -- References -- Chapter Seven: Functional assessment of N-terminal acetyltransferase variants -- 1 Introduction -- 2 Mutagenesis and cellular expression of NAT variants -- 2.1 Materials and equipment -- 2.2 Site-directed mutagenesis -- 2.3 Cell transfection, harvesting and lysis -- 2.4 Notes -- 3 Nt-specific antibody-based assay -- 3.1 Materials and equipment -- 3.2 Antibodies -- 3.3 SDS-PAGE and Western blot analysis -- 3.4 Notes -- 4 Immunoprecipitation (IP)-based acetylation (Ac)-assay -- 4.1 Materials and equipment -- 4.2 Immunoprecipitation -- 4.3 N-terminal acetylation assay -- 4.4 Western blot quantification -- 4.5 Notes -- 5 Summary and conclusion -- References -- Chapter Eight: A pan-N-formylmethionine-specific antibody as a tool for analyzing Nα-terminal formylation -- 1 Introduction -- 2 The challenge of analyzing Nt-formylation -- 3 Development of anti-pan-N-fMet-specific antibody using a single antigen -- 3.1 Antigen design -- 3.2 Antibody production -- 3.3 Antibody purification -- 3.3.1 Equipment, materials, and buffer recipes -- 3.3.2 Cell extract preparation -- 3.3.3 Resin preparation -- 3.3.4 Antibody selection -- 3.3.5 Antibody purification -- 3.4 Antibody qualification via SPOT array -- 3.4.1 Equipment, materials, and buffer recipes.
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| 955 |
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|a Elsevier ScienceDirect 2026-2027
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| 994 |
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|a 92
|b TXA
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|a Texas A&M University
|b College Station
|c Electronic Resources
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|h Library of Congress classification
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| 998 |
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