2-D Proteome Analysis ProtocolsAndrew J. Link With the completion of sequencing projects and the advancement of a- lytical tools for protein identification, proteomics—the study of the expressed part of the genome—has become a major region of the burgeoning field of functional genomics. High-resolution 2-D gels can reveal virtually all p- teins present in a cell or tissue at any given time, including posttranslationally modified proteins. Changes in the expression and structure of most cellular proteins caused by differentiation or external stimuli can be displayed and eventually identified using 2-D protein gels. 2-D Proteome Analysis Protocols covers all aspects of the use of 2-D protein electrophoresis for the analysis of biological problems. The contri- tors include many of the leaders in the fields of biochemistry and analytical chemistry who were instrumental in the development of high-resolution 2-D gels, immobilized pH gradients, computer analysis, and mass spectromet- based protein identification methodologies. This book is intended as a benchtop manual and guide both for novices to 2-D gels and for those aficionados who wish to try the newer techniques. Any group using protein biochemistry—especially in the fields of molecular biology, biochemistry, microbiology, and cell biology—should find this book eminently useful. 2-D Proteome Analysis Protocols takes the researcher through the c- plete process of working with 2-D protein gels from making the protein - tract to finally identifying the proteins of interest. It includes protocols for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeast, nematode, Drosophila, plants, mouse, and human. |
Contents
9 | |
Preparing 2D Protein Extracts from Yeast | 32 |
Eukaryotic Cell Labeling and Preparation for | 49 |
Fractionated Extraction of Total Tissue Proteins from Mouse | 67 |
Preparation and Solubilization of Body Fluids for | 87 |
Measuring the Radioactivity of 2D Protein Extracts | 105 |
Dimension IEF | 111 |
Nonequilibrium pH Gel Electrophoresis NEPHGE | 129 |
Coomassie Blue | 307 |
Detection of Total Proteins on Western Blots | 319 |
Protein Detection Using Reversible Metal Chelate Stains | 331 |
Gylcoprotein Detection of 2D Separated Proteins | 341 |
Image Acquisition in 2D Electrophoresis | 353 |
Computer Analysis of 2D Images | 363 |
Appel Amos Bairoch and Denis F Hochstrasser | 383 |
Comparing 2D Electrophoresis Gels Across Internet Databases | 393 |
LargeGel 2D Electrophoresis | 147 |
Advantages of Immobilized pH Gradients | 173 |
Analytical IPGDalt | 189 |
IPGDalt of Very Alkaline Proteins | 197 |
Running Preparative Carrier Ampholyte and Immobilized | 211 |
InGel Sample Rehydration of Immobilized pH Gradient | 221 |
Horizontal SDSPAGE for IPGDalt | 235 |
Casting and Running Vertical SlabGel Electrophoresis | 245 |
Nonreducing 2D Polyacrylamide Gel Electrophoresis | 255 |
Joan Goverman | 265 |
Hikaru Nagahara Robert R Latek Sergei A Ezhevsky | 271 |
Internal Standards for | 281 |
Kelvin H Lee and Michael G Harrington | 291 |
Appel Christine Hoogland Amos Bairoch | 411 |
Absolute Quantitation of 2D Protein Spots | 417 |
Identifying 2D Spots | 423 |
Barbara Magi Luca Bini Barbara Marzocchi | 431 |
Chloroformate | 445 |
NTerminal Amino Acid Sequencing of 2DE Spots | 461 |
Characterizing Proteins from 2DE Gels by Internal | 467 |
Identification of Proteins by MatrixAssisted Laser | 487 |
Sample Preparation Methods for Mass Spectrometric Peptide | 513 |
Protein Identification and Analysis Tools in the ExPASy Server | 531 |
Automated Protein Identification Using Microcolumn Liquid | 553 |
Peptide Sequencing of 2DE GelIsolated Proteins | 571 |
Other editions - View all
Common terms and phrases
2-D electrophoresis 2-D gel 2-D Proteome Analysis A. J. Link acetic acid acrylamide aliquots amino acid ammonium persulfate ampholytes Anal Biochem blotting bromophenol blue carrier ampholytes cassette cell centrifuge concentration Coomassie database deionized water detection digestion dissolved electroblotted extraction gel electrophoresis gel images gel solution glass plates Humana Press Inc IEF gel Immobiline immobilized pH gradients in-gel Incubate IPG gel strips labeled loaded MALDI mass spectrometry mass spectrum matching matrix membrane methanol Methods in Molecular mixture mol-wt Molecular Biology nanoelectrospray nitrocellulose Note patterns pellet peptide mass Pharmacia polyacrylamide gels polymerization preparation protein identification protein sequence protein spots Proteome Proteome Analysis Protocols PVDF reagent rehydration remove rinse room temperature sample SDS-PAGE Select sensitivity separation sequence tag silver staining solubilization Subheading supernatant SWISS-2DPAGE SWISS-PROT tandem mass teins tion tissue transfer two-dimensional electrophoresis two-dimensional gel two-dimensional gel electrophoresis urea volume Wash