The structure of a protein is essential for its function. Therefore, a variety of methods for analyzing protein structure have been developed recently; the most advanced and sensitive techniques have been selected for this manual. The presented procedures are well established and have been found useful in many laboratories; they are easy to perform and thus also suitable for beginners. Enclosed are protocols on the separation of proteins and peptides, manual and automated microsequencing, electrophoresis and blotting, analysis of amino acids, identification of cysteine residues and lipids as well as on mass spectrometry crystallization of macromolecules.

Klappentext

"Protein Structure Analysis - Preparation and Characterization" is a compilation of practical approaches to the structural analysis of proteins and peptides. Here, about 20 authors describe and comment on techniques for sensitive protein purification and analysis. These methods are used worldwide in biochemical and biotechnical research currently being carried out in pharmaceu­ tical and biomedical laboratories or protein sequencing facilities. The chapters have been written by scientists with extensive ex­ perience in these fields, and the practical parts are well documen­ ted so that the reader should be able to easily reproduce the described techniques. The methods compiled in this book were demonstrated in student courses and in the EMBO Practical Course on "Microsequence Analysis of Proteins" held in Berlin September 10-15, 1995. The topics also derived from a FEBS Workshop, held in Halkidiki, Thessaloniki, Greece, in April, 1995. Most of the authors participated in these courses as lecturers and tutors and made these courses extremely lively and successful. Since polypeptides greatly vary depending on their specific structure and function, strategies for their structural analysis must for the most part be adapted to each individual protein. Therefore, advantages and limitations of the experimen­ tal approaches are discussed here critically, so that the reader becomes familiar with problems that might be encountered.

Inhalt

I Separation of Proteins.- 1 Purification of Proteins for Sequencing.- 2 Affinity Chromatography of Proteins.- 3 Protein Purification by Aqueous Two-Phase Systems.- 4 Rapid HPLC Separation of Proteins, Peptides, and Amino Acids Using Short Columns Filled with Nonporous Silica-Based Particles.- II Separation of Peptides.- 5 Enzymatic and Chemical Cleavages of Proteins.- 6 Separation of Peptides Using HPLC and TLC Fingerprints.- 7 Microseparation of In Situ Digested Peptides.- 8 Isolation of Peptides for Microsequencing by In-Gel Proteolytic Digestion.- III Manual and Automated Microsequencing Methods.- 9 Automated Microsequencing: Introduction and Overview.- 10 A Manual Method for Protein Sequence Analysis Using the DABITC/PITC Method.- 11 Solid-Phase Sequencing of Peptides and Proteins.- 12 Sequence Analysis of the NH2-Terminally Blocked Proteins Immobilized on PVDF Membranes from Polyacrylamide Gels.- IV Electrophoresis and Blotting of Proteins.- 13 Two-Dimensional Electrophoresis.- 14 Semi-Dry Blotting onto Hydrophobic Membranes.- V Analysis of Amino Acids.- 15 Highly Sensitive Amino Acid Analysis.- 16 Quantitative Analysis of D- and L-Amino Acids by HPLC.- VI Identification of Cysteine Residues and Lipids in Proteins.- 17 Identification of Cysteine Residues and Disulfide Bonds in Proteins.- 18 Lipid Modifications of Proteins.- VII Mass Spectrometry in Peptide and Protein Analysis.- 19 Mass Spectrometry in Peptide and Protein Sequence Analysis.- 20 Carboxy-Terminal Sequencing by Combining Carboxypeptidase Y and P Digestion with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.- 21 Crystallization of Biological Macromolecules.