Basic Methods for the Biochemical Lab

262 Pages·2006·2.22 MB·English

by  Dr. Martin Holtzhauer (auth.)

Springer Labor Manual
Martin Holtzhauer
Basic Methods for
the Biochemical Lab
FirstEnglishEdition
23Figuresand86Tables
123
Dr.MartinHoltzhauer
HumanGmbHBranchIMTEC
Robert-Rössle-Strasse10
13125Berlin
Germany
e-mail:[email protected]
ISBN3-540-19267-0 1stGermanedition Springer-VerlagBerlinHeidelbergNewYork1988
ISBN3-540-58584-2 2ndGermanrevisededition Springer-VerlagBerlinHeidelbergNewYork1995
ISBN3-540-62435-X 3rdGermanrevisededition Springer-VerlagBerlinHeidelbergNewYork1997
LibraryofCongressControlNumber:2006922621
ISBN-10 3-540-32785-1 SpringerBerlinHeidelbergNewYork
ISBN-13 978-3-540-32785-1 SpringerBerlinHeidelbergNewYork
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ForDorothea,
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Preface
More than 20 years ago I started a collection of adapted protocols modified for spe-
cial applications and checked for daily usage in the biochemical (protein) lab. Small
“methods”withinlargepapersorpartsofchaptersinspecialbooks,overloadedwith
theoreticalexplanations,werethebasis.Myimaginationwasacookbook:Eachprotocol
containsalistofingredientsandashortinstruction(sometimesIwasnotveryconse-
quent,Ibegyourpardon!).Iproposedthisideatosomepublishinghouses,andin1988
Springer-Verlag published the first edition of Biochemische Labormethoden. Interest
andsuggestionsofnumerouscolleaguesledtoasecondandthirdGermanedition,and
nowthereseemstobeaninterestoutsideGermany,too.Thecontentsandformofthis
cookbookareperhapshelpfulforstudents,technicians,andscientistsinbiochemistry,
molecularbiology,biotechnology,andclinicallaboratory.
Startingfromthefirstedition,theaimofthisbookhasbeentoprovidesupporton
thebenchandastimulationofuser’smethodologicalknowledge,resultinginapossible
qualificationofhis/herexperimentalrepertoireand,asaspecialrequestforthereader
ofthisbook,animprovementofthe“basicprotocols.”
DuringmyprofessionallifeIhavereceivedinnumerablehintsandspecialtipsfrom
amultitudeofcolleaguesandco-workers.Theirknowledgeisnowpartofthepresent
protocolsandIgivemythankstothem.
I especially acknowledge Mrs. Susanne Dowe, because without her support and
helpfulcriticism,Ineverwouldhavetriedtomakeafurthereditionoftheseprotocols.
Berlin,January2006 MartinHoltzhauer
TableofContents
Abbreviations.......................................................... XVII
1 QuantitativeMethods................................................. 1
1.1 QuantitativeDeterminationsofProteins............................ 1
1.1.1 LowryProteinQuantification ............................. 2
1.1.1.1 StandardProcedure ............................. 2
1.1.1.2 ModificationbySargent ........................ 3
1.1.1.3 MicromethodonMicrotestPlates ................. 4
1.1.1.4 ProteinDeterminationinthePresence
ofInterferingSubstances ......................... 5
1.1.2 BradfordProteinDetermination.......................... 6
1.1.3 ProteinDeterminationinSDS-PAGESampleSolutions........ 7
1.1.4 ProteinDeterminationUsingAmidoBlack .................. 8
1.1.5 BCAProteinDetermination ............................... 9
1.1.5.1 BCAStandardProcedure......................... 9
1.1.5.2 BCAMicromethod .............................. 9
1.1.6 KjeldahlProteinDetermination .......................... 10
1.1.7 UVPhotometricAssayofProteinConcentration ............. 11
1.2 QuantitativeDeterminationofNucleicAcids........................ 13
1.2.1 SchmidtandThannhauserDNA,RNA,
andProteinSeparationProcedure.......................... 13
1.2.2 OrcinRNA(Ribose)Determination ........................ 14
1.2.3 DiphenylamineDNA(Deoxyribose)Determination .......... 14
1.2.4 QuantitativeDNADeterminationwithFluorescentDyes ...... 15
1.2.5 DeterminationofNucleicAcidsbyUVAbsorption ........... 16
1.3 QuantitativePhosphateDeterminations ............................ 17
1.3.1 DeterminationofInorganicPhosphateinBiologicSamples.... 17
1.3.2 DeterminationofTotalPhosphate .......................... 18
1.3.3 PhospholipidDetermination .............................. 18
1.4 MonosaccharideDetermination ................................... 19
1.5 CalculationsinQuantitativeAnalysis .............................. 20
2 Electrophoresis ...................................................... 23
2.1 PolyacrylamideGelElectrophoresisSystems ........................ 23
2.1.1 LaemmliSDS-PolyacrylamideGelElectrophoresis ........... 26
2.1.2 SDS-Polyacrylamide Gel Electrophoresis at Neutral pH
(NuPAGE)............................................... 31
X TableofContents
2.1.3 SDS-PolyacrylamideGelElectrophoresisAccording
toWeber,Pringle,andOsborn .......................... 32
2.1.4 Urea-SDS-PolyacrylamideGelElectrophoresis
fortheSeparationofLowMolecularWeightProteins ......... 34
2.1.5 TRICINE-SDS-PolyacrylamideGelElectrophoresis
forProteinsandOligopeptidesintheRange
of1000–50000Daltons.................................... 35
2.1.6 SDS-PolyacrylamideGelElectrophoresisatpH2.4............ 36
2.1.7 Urea-PolyacrylamideGelElectrophoresisforBasicProteins
atpH2.................................................. 37
2.1.8 AnodicDiscontinuousPolyacrylamideGelElectrophoresis
(NativePAGE) ........................................... 38
2.1.9 CathodicDiscontinuousPolyacrylamideGelElectrophoresis
(NativePAGE) ........................................... 39
2.1.10 AffinityElectrophoresis................................... 40
2.1.11 Two-Dimensional Polyacrylamide Gel Electrophoresis
(2D-PAGE;IEFfollowedbySDS-PAGE) ..................... 41
2.1.11.1 FirstDimension:IsoelectricFocusing(IEF)......... 42
2.1.11.2 SecondDimension:SDS-PAGE
(AcrylamideGradientGel) ....................... 44
2.2 AgaroseandPaperElectrophoresis ................................ 45
2.2.1 Non-denaturatingNucleicAcidElectrophoresis.............. 45
2.2.2 DenaturatingNucleicAcidElectrophoresis .................. 46
2.2.3 IdentificationofPhosphoaminoAcids(PaperElectrophoresis). 48
2.3 AidinElectrophoresis ........................................... 49
2.3.1 MarkerDyesforMonitoringElectrophoresis................. 49
2.3.1.1 AnodicSystems ................................. 49
2.3.1.2 CathodicSystems ............................... 49
2.3.2 MarkerProteinsforthePolyacrylamideGelElectrophoresis ... 50
2.3.3 CovalentlyColoredMarkerProteins ........................ 52
2.4 StainingProtocols ............................................... 53
2.4.1 StainingwithOrganicDyes................................ 53
2.4.1.1 AmidoBlack10B ............................... 54
2.4.1.2 CoomassieBrilliantBlueR250andG250 ........... 54
2.4.1.3 CoomassieBrilliantBlueR250Combined
withBismarckBrownR .......................... 55
2.4.1.4 FastGreenFCF ................................. 55
2.4.1.5 StainsAll....................................... 56
2.4.1.6 StainingofProteolipids,Lipids,andLipoproteins.... 56
2.4.2 SilverStainingofProteinsinGels .......................... 56
2.4.2.1 Citrate/FormaldehydeDevelopment ............... 57
2.4.2.2 AlkalineDevelopment ........................... 58
2.4.2.3 SilverStainingUsingTungstosilicicAcid ........... 58
2.4.2.4 SilverStainingofProteins:FormaldehydeFixation .. 59
2.4.2.5 SilverStainingofGlycoproteinsandPolysaccharides. 60
2.4.2.6 EnhancementofSilverStaining ................... 60
2.4.2.7 ReducingofSilver-StainedGels ................... 61
2.4.3 CopperStainingofSDS-PAGEGels ......................... 61
TableofContents XI
2.4.4 StainingofGlycoproteinsandPolysaccharidesinGels ........ 62
2.4.4.1 StainingwithSchiff’sReagent(PASStaining) ...... 62
2.4.4.2 StainingwithThymol............................ 63
2.4.5 StainingofBlottedProteinsonMembranes.................. 63
2.4.5.1 StainingonNitrocellulosewithDyes............... 63
2.4.5.2 StainingonNitrocellulosewithColloidalGold ...... 64
2.4.5.3 StainingonPVDFBlottingMembraneswithDyes ... 65
2.5 ElectroelutionfromGels ......................................... 66
2.5.1 PreparativeElectroelutionofProteins
fromPolyacrylamideGels ................................. 66
2.5.2 RemovalofSDS .......................................... 67
2.5.3 ElectrotransferofProteinsontoMembranes
(Electroblotting;WesternBlot):Semi-dryBlotting............ 68
2.5.4 ImmunochemicalDetectionofAntigensAfterElectrotransfer
(Immunoblotting)........................................ 70
2.5.4.1 DetectionUsingHorseradishPeroxidase(HRP) ..... 72
2.5.4.2 DetectionUsingAlkalinePhosphatase(AP) ........ 73
2.5.5 ChemiluminescenceDetectiononBlottingMembranes ....... 74
2.5.5.1 ChemiluminescenceUsingHRP................... 74
2.5.5.2 ChemiluminescenceUsingAP .................... 74
2.5.6 Carbohydrate-SpecificGlycoproteinDetection
AfterElectrotransfer...................................... 75
2.5.7 GeneralCarbohydrateDetectiononWesternBlots............ 76
2.5.8 AffinityBlotting.......................................... 77
2.5.9 TransferofNucleicAcids(SouthernandNorthernBlot) ..... 78
2.6 DryingofElectrophoresisGels .................................... 79
2.7 AutoradiographyofRadioactiveLabeledCompoundsinGels ......... 80
3 Chromatography..................................................... 83
3.1 Thin-LayerChromatography...................................... 83
3.1.1 IdentificationoftheN-terminalAminoAcidinPolypeptides
(TLCofModifiedAminoAcids............................. 83
3.1.2 Thin-LayerChromatographyofNucleosidePhosphates ....... 85
3.1.3 GradientThin-LayerChromatographyofNucleotides......... 85
3.1.4 IdentificationofPhosphatesonTLCPlates .................. 87
3.1.5 LipidExtractionandTLCofLipids ......................... 88
3.2 HintsforColumnChromatographyofProteins...................... 89
3.3 Gel Permeation Chromatography (GPC; Gel Filtration, GF;
Size-ExclusionChromatography,SEC) ............................. 93
3.3.1 SelectionofSupports ..................................... 96
3.3.2 FillingofaGelFiltrationColumn .......................... 97
3.3.3 SampleApplicationandChromatographicSeparation(Elution) 97
3.3.4 CleaningandStorage ..................................... 98
3.3.5 DeterminationofVoidVolumeV andTotalVolumeV ....... 99
0 t
3.3.6 RemovingofUnboundBiotinAfterConjugation
byGelFiltration(“Desalting”) ............................. 99
3.4 IonExchangeChromatography(IEC) .............................. 102
3.4.1 PreparationofIonExchangeSupports ...................... 103
XII TableofContents
3.4.2 CapacityTest ............................................ 104
3.4.3 SampleApplication....................................... 104
3.4.4 Elution ................................................. 105
3.4.5 CleaningandRegeneration................................ 105
3.4.6 High-PerformanceIonExchangeChromatography(HPIEC)
ofMono-andOligosaccharides ............................ 106
3.5 HydrophobicInteractionChromatography(HIC).................... 107
3.5.1 CapacityTest ............................................ 107
3.5.2 Elution ................................................. 108
3.5.3 Regeneration ............................................ 108
3.5.4 AnalyticalHPLCofHapten-ProteinConjugates .............. 108
3.6 AffinityChromatography(AC) .................................... 109
3.6.1 CyanogenBromideActivation
ofPolysaccharide-BasedSupports.......................... 113
3.6.1.1 DeterminationoftheDegreeofActivation.......... 114
3.6.2 CouplingtoCyanogenBromide-ActivatedGels............... 114
3.6.2.1 QuantitativeDeterminationofCoupledDiamine
Spacerswith2,4,6-TrinitrobenzeneSulfonicAcid.... 115
3.6.2.2 QuantitativeDeterminationofImmobilizedProtein . 116
3.6.2.3 ImmobilizationofWheatGermAgglutinin ......... 116
3.6.2.4 AffinityPurificationofHRP ...................... 117
3.6.2.5 AffinityChromatographyofImmunoglobulins
on Immobilized Antibodies (Immunoaffinity
Chromatography,IAC)........................... 117
3.6.2.6 AffinityChromatographyofRabbitIgG
onProtein-ASupports ........................... 118
3.6.3 ActivationofSepharosewithEpichlorohydrin ............... 119
3.6.3.1 DeterminationofEpoxyResidues ................. 119
3.6.4 ImmobilizationofMonosaccharides(Fucose)................ 119
3.6.5 ActivationwithDivinylsulfone............................. 120
3.6.6 CouplingofReactiveDyestoPolysaccharides(Dye-Ligand
Chromatography) ........................................ 121
3.6.7 CovalentCouplingofBiotin
(Biotin-Avidin/StreptavidinSystem)........................ 121
3.6.8 MetalChelateChromatography
ofProteinsContainingHis -Tag............................ 123
6
3.7 ConcentrationofDilutedProteinSolutions ......................... 124
3.7.1 AcidicPrecipitation ...................................... 124
3.7.2 SaltingOut .............................................. 124
3.7.3 PrecipitationUsingOrganicSubstances ..................... 125
3.7.4 Lyophilization(FreezeDrying)............................. 126
3.7.5 Ultrafiltration............................................ 127
4 ImmunochemicalProtocols ........................................... 129
4.1 ConjugationofHaptens(Peptides)toCarrierProteins ............... 129
4.1.1 ActivationofProteinswithTraut’sReagentYieldingProteins
withAdditionalFreeSHGroups............................ 132
4.1.2 ConjugationofMCA-GlyPeptidestoSH-CarryingProteins.... 132
TableofContents XIII
4.1.3 ConjugationofSulfhydrylPeptidesUsing
4-(N-Maleimidomethyl)-Cyclohexane-1-Carbonic Acid
N-HydroxysuccinimideEster(SMCC)....................... 133
β
4.1.4 -Galactosidase-ImmunoglobulinConjugate
(CouplingviaSHGroups) ................................. 134
β
4.1.4.1 EnzymeReactionof -Galactosidase............... 134
4.1.5 CarbodiimideCouplingofPeptidestoCarrierProteins
with1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide
(EDAC,EDC) ............................................ 134
4.1.6 ConjugationofHorseradishPeroxidase(Glycoproteins)
byPeriodateOxidation ................................... 135
4.1.7 ConjugationofPeptidestoCarrierProteins
UsingGlutaraldehyde(Two-StepProcedure)................. 136
4.1.8 ConjugationofHRPtoAntibodieswithGlutaraldehyde....... 137
4.1.9 Alkaline Phosphatase-Immunoglobulin Conjugate
(GlutaraldehydeProtocol)................................. 138
4.1.9.1 EnzymaticReactionofAlkalinePhosphatase
fromCalfIntestine .............................. 138
4.1.10 LabelingofImmunoglobulinswithFluorescentDyes ......... 138
4.1.11 Protein-ColloidalGoldConjugates ......................... 141
4.1.11.1 PreparationofColloidalGoldSol.................. 141
4.1.11.2 AdsorptionofProteintoColloidalGold ............ 142
4.2 ImmunizationofLaboratoryAnimals.............................. 143
4.3 AmmoniumSulfateFractionationofImmunoglobulins .............. 144
4.4 RemovalofUnspecificImmunoreactivities ......................... 146
4.4.1 PreparationofTissuePowder(LiverPowder) ................ 148
4.5 PreparationofEggYolkIgYFraction............................... 148
4.6 AntibodyFragmentation ......................................... 149
(cid:1)
4.6.1 F(ab)2FragmentsfromIgG ............................... 149
(cid:1)
4.6.2 Fab Fragments(Rabbit) .................................. 150
4.6.3 FabFragments(Rabbit)................................... 150
4.7 HeidelbergerCurve(PrecipitinCurve)........................... 150
4.8 OuchterlonyDouble-RadialImmunodiffusion .................... 151
4.8.1 PurificationofAgar ...................................... 151
4.8.2 PreparationofSlides...................................... 151
4.8.3 Immunodiffusion ........................................ 152
4.8.4 VisualizationofthePrecipitinLines ........................ 152
4.9 ImmunoprecipitationofAntigens ................................. 153
4.10 Immunoelectrophoresis.......................................... 154
4.11 Counterelectrophoresis .......................................... 155
4.12 Dot-BlotAssay .................................................. 156
4.13 EnzymeImmunosorbentAssay(EIA,ELISA) ....................... 157
4.13.1 IndirectEIAwithHRPConjugate .......................... 158
4.13.2 DeterminationofEnzymeActivitybyELISA................. 159
4.13.3 IsotypeDeterminationbyEIA(APConjugate) ............... 160