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P4617 Sigma

Proteome Revisited: Theory and Practice of all Relevant Electrophoretic Steps

  •  ISBN-10 0-444-50526-1

  •  ISBN-13 978-0-444-50526-2

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Related Categories Biotechnology Books, Books and Software, Labware, Proteomics Books
publication info   P.G. Righetti, et al., Elsevier Science, 2001, 408 pp., hard cover
mfr. no.   Elsevier

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General description

This covers electrophoretic steps leading to proteome analysis, i.e. isoelectric focusing (including immobilized pH gradients), SDS-PAGE electrophoresis and finally two-dimensional maps. It is a collection of modern methodologies leading to successful fractionation, analysis and characterization of every polypeptide spot in 2-D map analysis. It includes chapters on sophisticated mass spectrometry developments and navigating important databases. It contains several as yet unpublished protocols, correcting some of the existing ones and showing the pitfalls and limitations of others.

Table of Contents

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Part I: Isoelectric Focusing: Fundamentals. Perspectives and Limits. Optimization of the Separation Process.

Part I.I: Isoelectric Focusing: Fundamentals. Introduction. Isoelectric Focusing: Principles and Historical Aspects. References. Electrolyte Dissociation in Water Solution. Simple Electrolytes. Stepwise and parallel dissociation schemes for a bivalent protolyte. Relative concentration of different protolyte forms for stepwise and parallel schemes. Hydrogen ions concentration and buffer capacity. Ionization coefficient. Isoelectric point. Mobility of protolyte molecule. Non additive sum for buffer capacity in case of stepwise dissociation. Non amphoteric compounds and buffer capacity in "isoprotic state". References. Dissociation of Polyvalent Electrolytes. Acid-base equilibria, macroscopic and microscopic constants. Dissociation schemes of a hybrid type. Proton transfer tautomerism. Schemes with independent dissociation. Titration curve modeling.......................................
Part I.II: Optimization of the Elctrophoretic Separation. Buffering Capacity. Buffer capacity and buffer resource. Buffer properties of solutions of proteins and nucleic acids. Biopolymers as titration agents. References. Optimization of Electrophoretic Separation. Optimization of electrophoretic separation using pH-charge relationship. Calculation of mobility vs. pH. Relative charge difference for two components to be separated. Dependence of mobility on molecular mass in free solution. Isoelectric buffers. The concept of "Normalized b/l ratio". References. Two Dimensional Methods. Two-dimensional electrophoresis. Other two-dimensional separations. Mobility versus pH curves. References. Limitations of the Method of Isoelectric Focussing. Ways of generating pH gradients. Natural and artificial pH gradients. Thermal pH gradients. External temperature field.........................
Part II: Methodology Conventional Isoelectric Focusing in Gel Slabs and Capillaries and Immobilized pH Gradients. Introduction brief historical survey. Conventional isoelectric focusing in amphoteric buffers. General considerations. The basic method. Applications and limitations. Specific advantages. Carrier ampholytes. Equipment. Electrophoretic equipment. Polymerization cassette. The polyacrylamide matrix. Reagents. Gel formulations. Choice of carrier ampholytes. Gel preparation and electrophoresis. Assembling the mould. Filling the mould. Gel polymerization. Sample loading and electrophoresis. General protein staining. Coomassie Blue G-250. ........................................ IPG Methodology. Casting an Immobiline gel. Reswelling dry Immobiline gels. Electrophoresis. Staining and pH measurements. Storage of the Immobiline chemicals. Mixed bed, CA-IPG gels. Trouble-shooting. Some analytical results with IPGs. Capillary isoelectric focusing (cIEF). General considerations. cIEF methodology. General guidelines for cIEF. Increasing the resolution by altering the slope of the pH gradient. On the problem of protein solubility at their pI. Assessment of pH gradient and pI values in cIEF. Separation of peptides and proteins by CZE in isoelectric buffers. General properties of amphoteric, isoelectric buffers. Examples of some separations of proteins in isoelectric buffers. Troubleshooting for CZE in isoelectric buffers......................................... Mass spectrometry in proteomics. MALDI-TOF mass spectrometry. ESI mass spectrometry. Nanoelectrospray mass spectrometry. Mass spectrometry for quantitative proteomics. Labeling before extraction. Labeling after extraction. Multidimensional chromatography coupled to mass spectrometry. Informatics and Proteome: .......................................

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