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This volume provides the most widely used protocols and techniques employed in lipopolysaccharide( LPS) transport studies. Chapters detail identification and analysis of essential genes and crucial mutations in LPS transport-related genes, LPS profile determination, LPS chemical analysis, protein-LPS interaction, and LPS visualization. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.Authoritative and cutting-edge, Lipopolysaccharide Transport: Methods and Protocols aims to deliver an overview on the multidisciplinary study of LPS transport and to identify new and innovative antibacterial strategies.
Genetic Strategies to Study Essential LPS Biogenesis Genes
Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysacchari​de Transport
Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa
Analyzing the Function of Essential Genes by Plasmid Shuffling
Functional Analysis of Proteins Involved in LPS Transport
Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia coli
Exploring the Topology of Cytoplasmic Membrane Proteins Involved in Lipopolysacchari​de Biosynthesis by in Silico and Biochemical Analyses
Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein–LPS Interactions
A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell
Mass Spectrometry Analysis of Dynamics and Interactions of the LPS Translocon LptDE
Affinity Purification and Coimmunoprecipit​ation of Transenvelope Protein Complexes in Gram-Negative Bacteria
A Bacterial Two-Hybrid System for In Vivo Assays of Protein-Protein Interactions and Drug Discovery
Detecting Lipoproteins Sneaking Out of the Lipopolysacchari​de Leaflet
Tools for Structural Analysis of LPS and LPS Transport Proteins
Dissecting Lipopolysacchari​de Composition and Structure by GC-MS and MALDI Spectrometry
Probing the Functional Interaction Interface of Lipopolysacchari​de and Antimicrobial Peptides:​ A Solution-State NMR Perspective
Cryo-EM Analysis of the Lipopolysacchari​de Flippase MsbA
Protein Crystallization of Two Recombinant Lpt Proteins
Microscopy-Based Approaches
Metabolic Incorporation of Azido-Sugars into LPS to Enable Live-Cell Fluorescence Imaging
Use of Atomic Force Microscopy to Characterize LPS Perturbations