Electrophoresis in Practice
A Guide to Methods and Applications of DNA and Protein Separations
5. Auflage
Erschienen am 9. März 2016
XVI, 458 Seiten
978-3-527-69519-5 (ISBN)
Beschreibung
This fifth edition of the successful, long-selling classic has been completely revised and expanded, omitting some topics on obsolete DNA electrophoresis, but now with updated and extended methodical background sections.
The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.
The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.
Weitere Details
Weitere Ausgaben
Person
Reiner Westermeier worked after PhD graduation and Post-doc at the Technische Universität München for 30 years as a specialist for electrophoresis methods for leading bioanalytics and biotechnology companies. His area of responsibility included co-operation in product development, writing of scientic papers and method instructions, trouble shooting in customer laboratories, performing seminars and practical courses on electrophoresis and proteomics, as well as giving scientific talks at congresses on a world-wide basis. He is editor and author of several books, e.g. 'Electrophoresis in Practice' (in German and in English), 'Proteomics in Practice', and 'Difference Gel Electrophoresis'.
Inhalt
Contents
Part I
Fundamentals and History
Introduction
1 Electrophoresis
1.0 General
1.1 Electrophoresis in non-restrictive gels
1.1.1 Agarose gel electrophoresis
1.1.2 Polyacrylamide gel electrophoresis of low-molecular weight substances
1.2 Electrophoresis in restrictive gels
1.2.1 The Ferguson plot
1.2.2 Agarose gel electrophoresis
1.2.3 Polyacrylamide gel electrophoresis of nucleic acids
1.2.4 Polyacrylamide gel electrophoresis of proteins
2 Isotachophoresis
2.1 Migration with the same speed
2.2 "Ion train" separation
2.3 Zone sharpening effect
2.4 Concentration regulation effect
3 Isoelectric focusing
3.1 Principles
3.2 Gels for IEF
3.3 Temperature
3.4 Controlling the pH gradient
3.5 The kinds of pH gradients
3.5.1 Free carrier ampholytes
3.5.2 Immobilized pH gradients
3.6 Protein detection in IEF gels
3.7 Preparative isoelectric focusing
3.8 Titration curve analysis
4 Blotting
4.1 Principle
4.2 Transfer methods
4.3 Blotting membranes
4.4 Buffers for electrophoretic transfers
4.5 General staining
4.6 Blocking
4.7 Specific detection
4.8 Protein sequencing
4.9 Transfer problems
5 Interpretation of electropherograms
5.1 Introduction
5.1.1 Purity control
5.1.2 Quantification prerequisites
5.2 Image analysis
5.2.1 Hardware for image analysis
5.2.2 Software for image analysis
6 Proteome Analysis
6.1 General
6.2 Sample preparation
6.3 Two-dimensional electrophoresis
6.4 Detection techniques
6.5 Image analysis
6.6 Protein spot identification
6.6.1 Mass spectrometry methods
6.6.2 Peptide mass fingerprinting
6.6.3 Protein characterization
6.7 Bioinformatics
6.8 Functional proteomics
7 Instrumentation
7.1 Current and voltage conditions
7.2 Power supply
7.3 Separation chambers
7.3.1 Vertical apparatus
7.3.2 Horizontal apparatus
7.4 Staining apparatus for gels and blots
7.5 Automated electrophoresis
7.6 Instruments for 2-D electrophoresis
7.6.1 Isoelectric focusing apparatus
7.6.2 Multiple slab gel apparatus
7.7 Safety measures
7.8 Environmental aspects
Equipment for Part II
Instrumentation
Laboratory equipment
Consumables
Chemicals
Part II
Methods
Method 1: PAGE of dyes
1 Sample preparation
2 Stock solutions
3 Preparing the casting cassette
4 Casting the ultrathin-layer gels
5 Electrophoretic separation
Method 2: PAGE of DNA Fragments
1 Stock solutions
2 Preparing the gels
3 Sample preparation
4 Electrophoresis
5 Silver staining
Method 3: Agarose and immuno electrophoresis
1 Sample preparation
2 Stock solutions
3 Preparing the gels
4 Electrophoresis
5 Protein detection
Method 4: Titration curve analysis
1 Sample preparation
2 Stock solutions
3 Preparing the blank gels
4 Titration curve analysis
5 Coomassie and silver staining
6 Interpreting the curves
Method 5: Native PAGE in amphoteric buffers
1 Sample preparation
2 Stock solutions
3 Preparing the empty gels
4 Electrophoresis
5 Coomassie and silver staining
Method 6: Agarose IEF
1 Sample preparation
2 Preparing the agarose gel
3 Isoelectric focusing
5 Protein detection
Method 7: PAGIEF in rehydrated gels
1 Sample preparation
2 Stock solutions
3 Preparing the blank gels
4 Isoelectric focusing
5 Coomassie and silver staining
6 Perspectives
Method 8: Horizontal SDS-PAGE
1 Sample preparation
2 Stock solutions for the preparation of gels
3 Preparing the casting cassette
4 Gradient gel
5 Electrophoresis
6 Protein detection
7 Blotting
8 Perspectives
Method 9: Vertical PAGE
1 Sample preparation
2 Stock solutions
3 Single gel casting
4 Multiple gel casting
5 Electrophoresis
6 SDS electrophoresis of small peptides
7 Two-dimensional electrophoresis
8 DNA electrophoresis
9 Long shelflife gels
10 Pro
2D electrophoresis two-dimensional electrophoresis A ampere acc. according A,C,G,T adenine, cytosine, guanine, thymine ACES N-2-acetamido-2-aminoethanesulfonic acid AEBSF aminoethyl benzylsulfonyl fluoride AFLP amplified restriction fragment length polymorphism API atmospheric pressure ionization APS ammonium persulfate ARDRA amplified ribosomal DNA restriction analysis AU absorbance units 16-BAC benzyldimethyl-n-hexadecylammonium chloride BAC bisacryloylcystamine Bis N,N´-methylenebisacrylamide BNE blue native electrophoresis bp base pair BSA bovine serum albumin C crosslinking factor (%) CA carbonic anhydrase CAF chemically assisted fragmentation CAM coanalytical modification CAPS 3-(cyclohexylamino)-propanesulfonic acid CCD charge-coupled device CHAPS 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate CE capillary electrophoresis CID collision induced dissociation conc. concentrated CM carboxylmethyl CN-PAGE clear native page const. constant CTAB cetyltrimethylammonium bromide Da dalton DAF DNA amplification fingerprinting DBM diazobenzyloxymethyl DEA diethanolamine DEAE diethylaminoethyl DGGE denaturing gradient gel electrophoresis DHB 2,5-dihydroxybenzoic acid DIGE difference gel electrophoresis Disc discontinuous DMSO dimethylsulfoxide DNA desoxyribonucleic acid DPT diazophenylthioether dsDNA double stranded DNA DSCP double strand conformation polymorphism DTE dithioerythritol DTT dithiothreitol E field strength in volt per centimeter EDTA ethylenediaminetetraacetic acid ESI electro spray ionization EST expressed sequence tag FT-ICR Fourier transform - ion cyclotron resonance GC group specific component GMP good manufacturing practice h hour HED hydroxyethyldisulfide HEPES N-2-hydroxyethylpiperazine-N´-2-ethananesulfonic acid HMW high molecular weight HPCE high performance capillary electrophoresis HPLC high performance liquid chromatography I current in ampere, milliampere ICPL isotope-coded protein labeling IEF isoelectric focusing IgG immunoglobulin G IPG immobilized pH gradients ITP isotachophoresis kB kilobases kDa kilodaltons KR retardation coefficient LED light emitting diode LIF laser induced fluorescence LMW low molecular weight M mass mA milliampere MALDI matrix assisted laser desorption ionization MCE microchip electrophoresis MEKC micellar electrokinetic chromatography MES 2-(N-morpholino)ethanesulfonic acid min minute mol/L molecular mass MOPS 3-(N-morpholino)propanesulfonic acid mr relative electrophoretic mobility mRNA messenger RNA MS mass spectrometry Msn mass spectrometry with n mass analysis experiments MS/MS tandem mass spectrometry MW molecular weight NAP nucleic acid purifier Nonidet nonionic detergent NEPHGE non equilibrium pH gradient electrophoresis NHS N-hydroxy-succinimide O.D. optical density P power in watt p.a. per analysis PAG polyacrylamide gel PAGE polyacrylamide gel electrophoresis PAGIEF polyacrylamide gel isoelectric focusing PBS phosphate buffered saline PCR polymerase chain reaction PEG polyethylene glycol PFG pulsed field gel (electrophoresis) PGM phosphoglucose mutase pI isoelectric point PI protease inhibitor pK dissociation constant PMSF phenylmethyl-sulfonyl fluoride PPA piperidino propionamide PSD postsource dissociation (decay) PTM posttranslational modification PVC polyvinylchloride PVDF polyvinylidene difluoride r molecular radius RAPD random amplified polymorphic DNA REN rapid efficient nonradioactive Rf value relative distance of migration RFLP restriction fragment length polymorphism Rm relative electrophoretic mobility RNA ribonucleic acid RPA ribonuclease protection assay RuBP ruthenium II tris-bathophenantroline disulfonate s second SDS sodium dodecyl sulfate SNP single nucleotide polymorphism ssDNA single stranded DNA T total acrylamide concentration [%] TBE tris borate EDTA TBP tributyl phosphine TBS tris buffered saline TCA trichloroacetic...
Part I
Fundamentals and History
Introduction
1 Electrophoresis
1.0 General
1.1 Electrophoresis in non-restrictive gels
1.1.1 Agarose gel electrophoresis
1.1.2 Polyacrylamide gel electrophoresis of low-molecular weight substances
1.2 Electrophoresis in restrictive gels
1.2.1 The Ferguson plot
1.2.2 Agarose gel electrophoresis
1.2.3 Polyacrylamide gel electrophoresis of nucleic acids
1.2.4 Polyacrylamide gel electrophoresis of proteins
2 Isotachophoresis
2.1 Migration with the same speed
2.2 "Ion train" separation
2.3 Zone sharpening effect
2.4 Concentration regulation effect
3 Isoelectric focusing
3.1 Principles
3.2 Gels for IEF
3.3 Temperature
3.4 Controlling the pH gradient
3.5 The kinds of pH gradients
3.5.1 Free carrier ampholytes
3.5.2 Immobilized pH gradients
3.6 Protein detection in IEF gels
3.7 Preparative isoelectric focusing
3.8 Titration curve analysis
4 Blotting
4.1 Principle
4.2 Transfer methods
4.3 Blotting membranes
4.4 Buffers for electrophoretic transfers
4.5 General staining
4.6 Blocking
4.7 Specific detection
4.8 Protein sequencing
4.9 Transfer problems
5 Interpretation of electropherograms
5.1 Introduction
5.1.1 Purity control
5.1.2 Quantification prerequisites
5.2 Image analysis
5.2.1 Hardware for image analysis
5.2.2 Software for image analysis
6 Proteome Analysis
6.1 General
6.2 Sample preparation
6.3 Two-dimensional electrophoresis
6.4 Detection techniques
6.5 Image analysis
6.6 Protein spot identification
6.6.1 Mass spectrometry methods
6.6.2 Peptide mass fingerprinting
6.6.3 Protein characterization
6.7 Bioinformatics
6.8 Functional proteomics
7 Instrumentation
7.1 Current and voltage conditions
7.2 Power supply
7.3 Separation chambers
7.3.1 Vertical apparatus
7.3.2 Horizontal apparatus
7.4 Staining apparatus for gels and blots
7.5 Automated electrophoresis
7.6 Instruments for 2-D electrophoresis
7.6.1 Isoelectric focusing apparatus
7.6.2 Multiple slab gel apparatus
7.7 Safety measures
7.8 Environmental aspects
Equipment for Part II
Instrumentation
Laboratory equipment
Consumables
Chemicals
Part II
Methods
Method 1: PAGE of dyes
1 Sample preparation
2 Stock solutions
3 Preparing the casting cassette
4 Casting the ultrathin-layer gels
5 Electrophoretic separation
Method 2: PAGE of DNA Fragments
1 Stock solutions
2 Preparing the gels
3 Sample preparation
4 Electrophoresis
5 Silver staining
Method 3: Agarose and immuno electrophoresis
1 Sample preparation
2 Stock solutions
3 Preparing the gels
4 Electrophoresis
5 Protein detection
Method 4: Titration curve analysis
1 Sample preparation
2 Stock solutions
3 Preparing the blank gels
4 Titration curve analysis
5 Coomassie and silver staining
6 Interpreting the curves
Method 5: Native PAGE in amphoteric buffers
1 Sample preparation
2 Stock solutions
3 Preparing the empty gels
4 Electrophoresis
5 Coomassie and silver staining
Method 6: Agarose IEF
1 Sample preparation
2 Preparing the agarose gel
3 Isoelectric focusing
5 Protein detection
Method 7: PAGIEF in rehydrated gels
1 Sample preparation
2 Stock solutions
3 Preparing the blank gels
4 Isoelectric focusing
5 Coomassie and silver staining
6 Perspectives
Method 8: Horizontal SDS-PAGE
1 Sample preparation
2 Stock solutions for the preparation of gels
3 Preparing the casting cassette
4 Gradient gel
5 Electrophoresis
6 Protein detection
7 Blotting
8 Perspectives
Method 9: Vertical PAGE
1 Sample preparation
2 Stock solutions
3 Single gel casting
4 Multiple gel casting
5 Electrophoresis
6 SDS electrophoresis of small peptides
7 Two-dimensional electrophoresis
8 DNA electrophoresis
9 Long shelflife gels
10 Pro
Abbreviations, Symbols, Units
2D electrophoresis two-dimensional electrophoresis A ampere acc. according A,C,G,T adenine, cytosine, guanine, thymine ACES N-2-acetamido-2-aminoethanesulfonic acid AEBSF aminoethyl benzylsulfonyl fluoride AFLP amplified restriction fragment length polymorphism API atmospheric pressure ionization APS ammonium persulfate ARDRA amplified ribosomal DNA restriction analysis AU absorbance units 16-BAC benzyldimethyl-n-hexadecylammonium chloride BAC bisacryloylcystamine Bis N,N´-methylenebisacrylamide BNE blue native electrophoresis bp base pair BSA bovine serum albumin C crosslinking factor (%) CA carbonic anhydrase CAF chemically assisted fragmentation CAM coanalytical modification CAPS 3-(cyclohexylamino)-propanesulfonic acid CCD charge-coupled device CHAPS 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate CE capillary electrophoresis CID collision induced dissociation conc. concentrated CM carboxylmethyl CN-PAGE clear native page const. constant CTAB cetyltrimethylammonium bromide Da dalton DAF DNA amplification fingerprinting DBM diazobenzyloxymethyl DEA diethanolamine DEAE diethylaminoethyl DGGE denaturing gradient gel electrophoresis DHB 2,5-dihydroxybenzoic acid DIGE difference gel electrophoresis Disc discontinuous DMSO dimethylsulfoxide DNA desoxyribonucleic acid DPT diazophenylthioether dsDNA double stranded DNA DSCP double strand conformation polymorphism DTE dithioerythritol DTT dithiothreitol E field strength in volt per centimeter EDTA ethylenediaminetetraacetic acid ESI electro spray ionization EST expressed sequence tag FT-ICR Fourier transform - ion cyclotron resonance GC group specific component GMP good manufacturing practice h hour HED hydroxyethyldisulfide HEPES N-2-hydroxyethylpiperazine-N´-2-ethananesulfonic acid HMW high molecular weight HPCE high performance capillary electrophoresis HPLC high performance liquid chromatography I current in ampere, milliampere ICPL isotope-coded protein labeling IEF isoelectric focusing IgG immunoglobulin G IPG immobilized pH gradients ITP isotachophoresis kB kilobases kDa kilodaltons KR retardation coefficient LED light emitting diode LIF laser induced fluorescence LMW low molecular weight M mass mA milliampere MALDI matrix assisted laser desorption ionization MCE microchip electrophoresis MEKC micellar electrokinetic chromatography MES 2-(N-morpholino)ethanesulfonic acid min minute mol/L molecular mass MOPS 3-(N-morpholino)propanesulfonic acid mr relative electrophoretic mobility mRNA messenger RNA MS mass spectrometry Msn mass spectrometry with n mass analysis experiments MS/MS tandem mass spectrometry MW molecular weight NAP nucleic acid purifier Nonidet nonionic detergent NEPHGE non equilibrium pH gradient electrophoresis NHS N-hydroxy-succinimide O.D. optical density P power in watt p.a. per analysis PAG polyacrylamide gel PAGE polyacrylamide gel electrophoresis PAGIEF polyacrylamide gel isoelectric focusing PBS phosphate buffered saline PCR polymerase chain reaction PEG polyethylene glycol PFG pulsed field gel (electrophoresis) PGM phosphoglucose mutase pI isoelectric point PI protease inhibitor pK dissociation constant PMSF phenylmethyl-sulfonyl fluoride PPA piperidino propionamide PSD postsource dissociation (decay) PTM posttranslational modification PVC polyvinylchloride PVDF polyvinylidene difluoride r molecular radius RAPD random amplified polymorphic DNA REN rapid efficient nonradioactive Rf value relative distance of migration RFLP restriction fragment length polymorphism Rm relative electrophoretic mobility RNA ribonucleic acid RPA ribonuclease protection assay RuBP ruthenium II tris-bathophenantroline disulfonate s second SDS sodium dodecyl sulfate SNP single nucleotide polymorphism ssDNA single stranded DNA T total acrylamide concentration [%] TBE tris borate EDTA TBP tributyl phosphine TBS tris buffered saline TCA trichloroacetic...
