Post-translational processing : a practical approach / edited by S.J. Higgins and B.D. Hames.

Includes bibliographical references and index.

Print version record.

Post - Translational Modification: A Practical Approach and its companion volume Protein Expression: A Practical Approach form the final part of the PAS mini-series on protein synthesis and processing. This volume begins with a chapter on protein sequencing followed by a chapter on protein folding and import into organelles. The next three chapters cover the three major forms of covalent modification: phosphorylation, glycosylation, and lipid modification. Proteolytic processing the is the next topic and the final two chapters are concerned with protein turnover in mammalian cells and yeast. This book is a comprehensive volume of the best current methodology and is designed to be used at the bench or away from the bench to gain insight into future experimental approached.

Intro; Contents; List of Contributors; Abbreviations; 1. Sequence analysis of expressed proteins; 1. Introduction; 2. N-terminal sequence analysis; Automated sequencing; 3. Sample preparation; Sample preparation by SDS-PAGE; Sample preparation by HPLC; Other procedures for sample preparation; 4. N-terminal blocking; Protein fragmentation; 5. C-terminal sequencing; Chemical analysis of the C-terminus; Enzymic analysis of the C-terminus; 6. Mass spectrometric analysis of proteins; A brief guide to mass spectrometers; Molecular mass determination; Sequencing by mass spectrometry; References

2. Protein folding and import into organelles1. Introduction; 2. Preparation of preprotein substrates for in vitro import into organelles; Preparation of mRNA for in vitro translation; Synthesis of preproteins in the reticulocyte lysate; Synthesis of preproteins in wheat germ lysate; Synthesis of preproteins in yeast cytosol; Synthesis of preproteins in Escherichia coli; Dihydrofolate reductase as a model protein for import studies; 3. Import of proteins into mitochondria; Isolation of mitochondria for import studies; Import of preproteins into isolated mitochondria

Generation of translocation intermediatesLocalization of imported proteins; 4. Import of proteins into microsomes (endoplasmic reticulum); Isolation of microsomes; Import of preproteins into microsomes; 5. Import of proteins into other organelles; 6. Analysis of protein import into organelles; Monitoring the association of proteins with membranes; Analysis of protein complexes involved in organelle import; Analysis of protein folding after import into organelles; Acknowledgements; References; 3. Analysis of protein phosphorylation; 1. Introduction

2. Investigating protein phosphorylation systems3. Phosphorylation of proteins in intact preparations; General considerations; Intact animals; Tissue slices; Isolated cells; 4. Phosphorylation of proteins in cell-free preparations; General considerations; Labelling and stimulation of cell-free preparations; 5. Analysis of phosphorylated proteins; General considerations; Quantification of phosphoproteins; Phosphoprotein separation; Protein isolation by immunomethods; Analysis of multisite phosphorylation; Phosphoamino acid analysis; Analysis of the state of phosphorylation

6. Analysis of protein kinasesGeneral considerations; Analysis of protein kinase activity in vitro; Analysis of specific protein kinases in vitro; Analysis of protein kinase activity in intact cells; Purification of protein kinases; 7. Phosphoprotein phosphatases; General considerations; P-Ser/P-Thr phosphoprotein phosphatases; Tyrosine-specific phosphoprotein phosphatases; References; 4. Protein glycosylation; 1. Introduction; Protein glycosylation; Oligosaccharide structures; Glycosylation pathways; Characterization of protein glycosylation; 2. Is my protein glycosylated?

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