Sheet3ProteinPurpose/AimMethodNature of MethodAgent/Instrument usedMechanismMode of AnalysisN-terminal Analysis Edmans DegradationChemicalPhenyisothiocyanateFormation of phenylhydantoin complex with amino terminal amino acidChromatographyC-terminal AnalysisCarboxypeptidasesEnzymaticCarboxypetidasePostively charged amino acid Arg together with a hydrophobic pocket increases preference to carboxy terminalChromatography and graphical.ConcentrationFolin Ciocalteau MethodChemicalPhosphomolybdate, phosphotungsatereduction of molybdate by tyrosine and phenylanaline, reduction of copper by proteinColorimetryFunctionKinetic and non-kinetic assaysChemical Vary with procedureVary with procedureVary with procedureCharge Ion-exchange ChromatographyChemicalcharged resinsFormation of ionic bond between resin and protein due to opposite charge and subsequent elutionColorimetrySizeGel filtrationChemicalAgarose and other polymeric gelsExclusion or inclusion of a protien through the cavity btween gel beadsChromatographyMolecular massSDS-PAGEChemicalSDS,Polyacrylamide,agaroseSeparation of protein based on shape and size into constiutent polypeptides, Mw established through markersElectro-analyticalMass SpectroscopyPhysicalAstons Mass SpectrophotometerMALDI.Ionization followed by fragmentation of ionized proteins. Fragements sorted according to their m/z ratio.PhysicalTertiary StructureX-ray CrystllographyPhysicalX-raysDiffraction of X-rays by atoms in crystallized protein with respect to their postions.Phyical Membrane ProteinsHydropathy plotsMathematicalNonePlot of position of amino acid and their hydrophobic indexGraphicalSecondary structureBioinformaticsPrimary Structure/SequencingEdmans DegradationQuartenery structureSDS-PAGEChemicalDetermination of no of subunits and their nature.ChemicalAmplification of proteinRDTBiologicalBacterial plasmidsPlasmid ligated with gene producing the protein of interest.BiologicalFoldingCircular dichroismPhysicalUltraviolet lightDifferential absorption of light by individual amino acids.SpectroscopicIsolation/SeparationElectrophoresisLocationEpitope-taggingBiochemicalGene of interest is modified to form an epitope attachd to the protein. Antibodies generated against epitope to identify locationDNAIsolationDifferential extractionChemicalEDTA, Ethanol,Chlorofrom, SDS, Proteinases etcElimination of each biomolecule without disturbing DNA. DNA spooled out and dissolved in buffer.ChemicalDetectionColorimetryPhysicalColorimeterDNA, double and single strande have characteristic absorbance at 260nm.ColorimetryDetectionAgarose electrophoresisChemicalEthidium BromideEthidium Bromide intercalted between DNA basepairs and fluoresces when exposed to UV light.ChemicalConcentrationColorimetryStandard concentration at a given absorbance to determine concentration of unknown sample of DNAColorimetryConcentrationColorimetryDiphenylamineDehydration of deoxyriboe of purines followed by conversion to levulylaldehyde which rects with DPA to given Blue color.ColorimetrySequenceSangers Dideoxy MethodBiochemicalDeoxynucleotides,dideoxynucleotides, DNA PolymeraseIncorparation dideoxynucleotides to growing DNA chain leading to chain termination. Electrophoresis to identify sequence.Chemical,Electro-analyticalAmplificationPCRBiochemicalAutomator, Taq Polymerase, dNTPs etc.Enzymatic polymeriation Of DNA followed by separation of strands which are furthur polymerized.EnzymaticIdentification of Gene of interestSouthern BlottingBiochemicalAgarose Gel, Nitocellulose membrane, DNA from source, ProbeComplimentary binding of Probe to corresponding DNA by base pairing.BiochemicalGene activityAffinity ChromatographyBiochemicalPoly-g tagged affinity resinsActive gene produce many m-RNA which agre affinity eluted with poly-G by complimentary base pairing.ChemicalNumber of exonsR-loop analysisPhysicalElectron microscopeFormation of R shaped loops in RNA-DNA hybrids due to splicing. No of exon=No of loops+1BiochemicalPresence of origin siteRDT, bioassayBiochemicalyeast cells,plasmids with suspected sequence and a markerYeast cells containg plasmid with origin multiply in cultures lacking the amino acid but carry a gene to synthesize it.BiochemicalHelical DensityEthidim Bromide electrophoresisChemicalEthidium bromide, DNA fragmentEthidium bromide intercaltes between DNA and unwinds DNA by one turn visualized in the gel.ChemicalConsensus sequenceSite directed mutagenesisChemicalMutagen, dnaMutation of a base in a gene followed by measurement of its activity for increase or decrease. ChemicalLocationIn-situ hybridizationChemical radiolabelled nucleotides, organism of interestReal time visualization of site of DNA or m-RNA sequence in cell by allowing the cell to make a labelled sequenceChemical