Basic Methods for the Biochemical Lab

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1 Springer Lab Manuals Basic Methods for the Biochemical Lab Bearbeitet von Martin Holtzhauer 1. Auflage Taschenbuch. xviii, 252 S. Paperback ISBN Format (B x L): 15,5 x 23,5 cm Gewicht: 416 g Weitere Fachgebiete > Chemie, Biowissenschaften, Agrarwissenschaften > Chemie Allgemein > Chemische Labormethoden, Stöchiometrie Zu Leseprobe schnell und portofrei erhältlich bei Die Online-Fachbuchhandlung beck-shop.de ist spezialisiert auf Fachbücher, insbesondere Recht, Steuern und Wirtschaft. Im Sortiment finden Sie alle Medien (Bücher, Zeitschriften, CDs, ebooks, etc.) aller Verlage. Ergänzt wird das Programm durch Services wie Neuerscheinungsdienst oder Zusammenstellungen von Büchern zu Sonderpreisen. Der Shop führt mehr als 8 Millionen Produkte.

2 Abbreviations... XVII 1 Quantitative Methods QuantitativeDeterminationsofProteins Lowry ProteinQuantification StandardProcedure Modification by Sargent MicromethodonMicrotestPlates Protein Determination in the Presence ofinterferingsubstances Bradford ProteinDetermination ProteinDeterminationinSDS-PAGESampleSolutions ProteinDeterminationUsingAmidoBlack BCAProteinDetermination BCAStandardProcedure BCAMicromethod Kjeldahl ProteinDetermination UVPhotometricAssayofProteinConcentration QuantitativeDeterminationofNucleicAcids Schmidt and Thannhauser DNA, RNA, andproteinseparationprocedure OrcinRNA(Ribose)Determination DiphenylamineDNA(Deoxyribose)Determination QuantitativeDNADeterminationwithFluorescentDyes DeterminationofNucleicAcidsbyUVAbsorption QuantitativePhosphateDeterminations Determination of Inorganic Phosphate in Biologic Samples DeterminationofTotalPhosphate PhospholipidDetermination MonosaccharideDetermination CalculationsinQuantitativeAnalysis Electrophoresis PolyacrylamideGelElectrophoresisSystems Laemmli SDS-PolyacrylamideGelElectrophoresis SDS-Polyacrylamide Gel Electrophoresis at Neutral ph (NuPAGE)... 31

3 X SDS-Polyacrylamide Gel Electrophoresis According to Weber, Pringle,andOsborn Urea-SDS-Polyacrylamide Gel Electrophoresis fortheseparationoflowmolecularweightproteins TRICINE-SDS-Polyacrylamide Gel Electrophoresis for Proteins and Oligopeptides in the Range of Daltons SDS-PolyacrylamideGelElectrophoresisatpH Urea-Polyacrylamide Gel Electrophoresis for Basic Proteins atph Anodic Discontinuous Polyacrylamide Gel Electrophoresis (NativePAGE) Cathodic Discontinuous Polyacrylamide Gel Electrophoresis (NativePAGE) AffinityElectrophoresis Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE;IEFfollowedbySDS-PAGE) FirstDimension:IsoelectricFocusing(IEF) Second Dimension: SDS-PAGE (AcrylamideGradientGel) AgaroseandPaperElectrophoresis Non-denaturatingNucleicAcidElectrophoresis DenaturatingNucleicAcidElectrophoresis Identification of Phosphoamino Acids (Paper Electrophoresis) AidinElectrophoresis MarkerDyesforMonitoringElectrophoresis AnodicSystems CathodicSystems Marker Proteins for the Polyacrylamide Gel Electrophoresis CovalentlyColoredMarkerProteins Staining Protocols Staining with Organic Dyes AmidoBlack10B CoomassieBrilliantBlueR250andG Coomassie Brilliant Blue R250 Combined withbismarckbrownr FastGreenFCF StainsAll Staining of Proteolipids, Lipids, and Lipoproteins Silver Staining of Proteins in Gels Citrate/FormaldehydeDevelopment Alkaline Development Silver Staining Using Tungstosilicic Acid Silver Staining of Proteins: Formaldehyde Fixation Silver Staining of Glycoproteins and Polysaccharides Enhancement of Silver Staining ReducingofSilver-StainedGels Copper Staining of SDS-PAGE Gels

4 XI Staining of Glycoproteins and Polysaccharides in Gels Staining with Schiff s Reagent (PAS Staining) Staining with Thymol Staining of Blotted Proteins on Membranes Staining on Nitrocellulose with Dyes Staining on Nitrocellulose with Colloidal Gold Staining on PVDF Blotting Membranes with Dyes ElectroelutionfromGels Preparative Electroelution of Proteins frompolyacrylamidegels RemovalofSDS Electrotransfer of Proteins onto Membranes (Electroblotting; Western Blot): Semi-dry Blotting Immunochemical Detection of Antigens After Electrotransfer (Immunoblotting) DetectionUsingHorseradishPeroxidase(HRP) Detection Using Alkaline Phosphatase (AP) ChemiluminescenceDetectiononBlottingMembranes ChemiluminescenceUsingHRP ChemiluminescenceUsingAP Carbohydrate-Specific Glycoprotein Detection AfterElectrotransfer GeneralCarbohydrateDetectiononWesternBlots AffinityBlotting Transfer of Nucleic Acids (Southern andnorthernblot) DryingofElectrophoresisGels AutoradiographyofRadioactiveLabeledCompoundsinGels Chromatography Thin-Layer Chromatography Identification of the N-terminal Amino Acid in Polypeptides (TLCofModifiedAminoAcids Thin-Layer Chromatography of Nucleoside Phosphates Gradient Thin-Layer Chromatography of Nucleotides IdentificationofPhosphatesonTLCPlates LipidExtractionandTLCofLipids HintsforColumnChromatographyofProteins Gel Permeation Chromatography (GPC; Gel Filtration, GF; Size-ExclusionChromatography,SEC) SelectionofSupports Filling of a Gel Filtration Column Sample Application and Chromatographic Separation (Elution) Cleaning and Storage Determination of Void Volume V 0 and Total Volume V t Removing of Unbound Biotin After Conjugation bygelfiltration( Desalting ) IonExchangeChromatography(IEC) PreparationofIonExchangeSupports

5 XII CapacityTest SampleApplication Elution Cleaning and Regeneration High-Performance Ion Exchange Chromatography (HPIEC) ofmono-andoligosaccharides HydrophobicInteractionChromatography(HIC) CapacityTest Elution Regeneration AnalyticalHPLCofHapten-ProteinConjugates AffinityChromatography(AC) Cyanogen Bromide Activation ofpolysaccharide-basedsupports DeterminationoftheDegreeofActivation Coupling to Cyanogen Bromide-Activated Gels Quantitative Determination of Coupled Diamine Spacers with 2,4,6-Trinitrobenzene Sulfonic Acid Quantitative Determination of Immobilized Protein Immobilization of Wheat Germ Agglutinin AffinityPurificationofHRP Affinity Chromatography of Immunoglobulins on Immobilized Antibodies (Immunoaffinity Chromatography,IAC) Affinity Chromatography of Rabbit IgG on Protein-A Supports ActivationofSepharosewithEpichlorohydrin DeterminationofEpoxyResidues Immobilization of Monosaccharides (Fucose) ActivationwithDivinylsulfone Coupling of Reactive Dyes to Polysaccharides (Dye-Ligand Chromatography) Covalent Coupling of Biotin (Biotin-Avidin/Streptavidin System) Metal Chelate Chromatography of Proteins Containing His 6 -Tag ConcentrationofDilutedProteinSolutions Acidic Precipitation SaltingOut PrecipitationUsingOrganicSubstances Lyophilization (Freeze Drying) Ultrafiltration Immunochemical Protocols ConjugationofHaptens(Peptides)toCarrierProteins Activation of Proteins with Traut s Reagent Yielding Proteins withadditionalfreeshgroups Conjugation of MCA-Gly Peptides to SH-Carrying Proteins

6 XIII Conjugation of Sulfhydryl Peptides Using 4-(N-Maleimidomethyl)-Cyclohexane-1-Carbonic Acid N-Hydroxysuccinimide Ester (SMCC) β-galactosidase-immunoglobulin Conjugate (Coupling via SH Groups) Enzyme Reaction of β-galactosidase Carbodiimide Coupling of Peptides to Carrier Proteins with 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDAC,EDC) Conjugation of Horseradish Peroxidase (Glycoproteins) byperiodateoxidation Conjugation of Peptides to Carrier Proteins UsingGlutaraldehyde(Two-StepProcedure) ConjugationofHRPtoAntibodieswithGlutaraldehyde Alkaline Phosphatase-Immunoglobulin Conjugate (GlutaraldehydeProtocol) Enzymatic Reaction of Alkaline Phosphatase fromcalfintestine Labeling of Immunoglobulins with Fluorescent Dyes Protein-Colloidal Gold Conjugates PreparationofColloidalGoldSol AdsorptionofProteintoColloidalGold Immunization of Laboratory Animals AmmoniumSulfateFractionationofImmunoglobulins RemovalofUnspecificImmunoreactivities PreparationofTissuePowder(LiverPowder) PreparationofEggYolkIgYFraction AntibodyFragmentation F(ab )2FragmentsfromIgG Fab Fragments(Rabbit) FabFragments(Rabbit) Heidelberger Curve(PrecipitinCurve) Ouchterlony Double-Radial Immunodiffusion PurificationofAgar Preparation of Slides Immunodiffusion Visualization of the Precipitin Lines ImmunoprecipitationofAntigens Immunoelectrophoresis Counterelectrophoresis Dot-BlotAssay EnzymeImmunosorbentAssay(EIA,ELISA) Indirect EIA with HRP Conjugate DeterminationofEnzymeActivitybyELISA IsotypeDeterminationbyEIA(APConjugate)

7 XIV 5 Centrifugation SpeedvsCentrifugalForceGraphs DifferentialCentrifugation DensityGradientCentrifugation Pre-formed Discontinuous Gradient Centrifugation: IsolationofLiverCellNuclei Sucrose Gradient Centrifugation: Preparation of Surface Membranes (Sarcolemma, SL) of Heart Muscle Cells Determination of a Marker Enzyme: Ouabain-Sensitive Na,K-ATPase Receptor Determination: DHP Binding Sites onsurfacemembranes Determination of the Dissociation andassociationkineticsofthedhpreceptors RNA Separation by Non-Denaturating Sucrose Density GradientCentrifugation DenaturatingRNAGradientCentrifugation IsopycnicCentrifugation Purification of High Molecular Weight DNA incsclgradients CellFractionationUsingPercoll PreparationofHumanLymphocytes Radioactive Labeling RadioactiveDecay Decay Tables for 32-Phosphorus, 35-Sulfur, and 125-Iodine Enzymatic [ 32 P]-PhosphateIncorporationintoProteins Iodination with [ 125 I]-Iodine Reagents Chloramine-TProtocol Iodination with Bolton Hunter Reagent Scintillation Cocktails for Liquid Scintillation Counting Buffers TheoreticalConsiderations PlotforBufferCalculations ph Indicators BufferRecipes CommonlyUsedBuffers Buffers and Media for Tissue and Cell Culture andorganperfusion phcalibrationbuffers VolatileBuffers Tables ConcentrationUnits ConversionFactorsforSIUnits DataofFrequentlyUsedSubstances ProteinData

8 XV 8.5 ProteaseInhibitors Single-LetterCodesandMolecularMassesofAminoAcids SpectroscopicDataofNucleotides Detergents( Surfactants ) RefractiveIndexandDensityofSucroseSolutions AmmoniumSulfateSaturationTable DilutedSolutions MixtureRule Statistics and Data Analysis StatisticalEquations MeanandRelatedFunctions Correlation:LinearRegression The t-test(student stest) DataAnalysis Receptor Ligand Binding EnzymeKinetics DeterminationofMolecularMassbySDS-PAGE DiagnosticSensitivityandSpecificity SoftwarefortheLab DataAnalysisandPresentation SoftwareforStatistics OtherSoftware SelectedInternetLinks Subject Index