Protein Chromatography

This book examines vital topics in protein biochemistry and presents methods involving the generation and purification of recombinant proteins through recombinant antibody production and the tagging of proteins, as well as variations on classic techniques.

A prerequisite for elucidating the structure and function of any protein is the prior purification of that protein. This necessity has led to the development of many purification schemes and chromatographic methods for the isolation of native proteins from complex sources. In Protein Chromatography: Methods and Protocols, leading researchers present clear protocol-style chapters that are suitable for newcomers and experts alike. The book opens with vital topics in protein biochemistry, addressing such areas as protein stability and storage, avoiding proteolysis during chromatography, protein quantitation methods including immuno-qPCR, and the contrasting challenges that microfluidics and scale-up production pose to the investigator, and then it segues into key methods involving the generation and purification of recombinant proteins through recombinant antibody production and the tagging of proteins, amongst other means, as well as many variations on classic techniques such as ion-exchange and immunoaffinity chromatography. Written in the highly successful Methods in Molecular Biology™ series format, protocols chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Protein Chromatography: Methods and Protocols will greatly aid scientists in establishing these essential techniques in their own laboratories and furthering our understanding of the many imperative functions of proteins.

Features contributions from experienced scientists with hands-on expertise in the field presenting step-by-step methodologies, tips, and associated explanatory notes Includes a large section on the generation and purification of recombinant proteins Addresses protein biochemistry with clear, step-by-step techniques Includes supplementary material: sn.pub/extras

Inhalt
A Digest of Protein Purification.- Gel-Filtration Chromatography.- Immunoaffinity Chromatography.- Avoiding Proteolysis During Protein Chromatography.- Scale-Up of Protein Purification: Downstream Processing Issues.- Phage Display: A Powerful Technology for the Generation of High Specificity Affinity Reagents from Alternative Immune Sources.- Engineering Protein Stability.- Microfluidics in Protein Chromatography.- Tagging Recombinant Proteins to Enhance Solubility and Aid Purification.- Storage and Lyophilization of Pure Proteins.- Differential Precipitation and Solubilization of Proteins.- Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase.- Protein Quantitation and Analysis of Purity.- Purification of Proteins Fused to Glutathione S-Tranferase.- Purification of Proteins Fused to Maltose-Binding Protein.- Purification of Proteins from Baculovirus-Infected Insect Cells.- Purification of Poly-Histidine-Tagged Proteins.- Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment of Phosphopeptides and Analysis by CID/ETD Tandem Mass Spectrometry.- Tag Removal by Site-Specific Cleavage of Recombinant Fusion Proteins.- Purification of Antibodies Using Affinity Chromatography.- Optimized Generation of High-Affinity, High-Specificity Single-Chain Fv Antibodies from Multi-Antigen Immunized Chickens.- Measuring Protein-Protein Interactions Using Biacore.- Ultra-Performance Liquid Chromatography-Mass Spectrometry of Proteins.- Hydrophobic Interaction Chromatography.- Fast Protein Liquid Chromatography.- Shotgun Proteomics: A Qualitative Approach Applying Isoelectric Focusing on Immobilized pH Gradient and LC-MS/MS.- Shotgun Proteomics: A Relative Quantitative Approach Using Off-Gel Electrophoresis and LC-MS/MS.- Clinical Proteomics: Liquid Chromatography-Mass Spectrometry (LC/MS) Purification Systems.- Strategies for the Purification of Membrane Proteins.- A Multi-Step ChromatographicStrategy to Purify Three Fungal Endo--Glucanases.