Western 杂交

PreparingProteinLysates

• PreparingProteinLysatesforWesternBlotting(AntisenseResearchGroup)
• YeastLysatesforWesterns(PMCI)
• ExtractsforYeastWesterns(HerskowitzLab
• PreparationofBrainMembraneFractionsforWesternBlot(BDPharMingen)

WesternBlotting

• SDS/PAGEProteinTransfer(GimilaLab)
• GeneralWesternBlotProtocol(KPL)ThisprocedurewaswrittenasageneralprotocolforusewithstandardTris-GlycineSDS-PAGEgelsandnitrocellulosemembranes.Itshouldgiveacceptableresultsinmostsituations.Optimizationforparticularsituationsmayrequiremodifyingtheconditionsdescribed.

·Southerns,Westerns,andNortherns:MolecularCloningTechniques(BrianWhite,MIT)Basictheory,definitionandprocedureswithillustration

·WesternBlotting(HowellLab)Proceduresforpreparationofcelllysate,gel,electrophoresis,transfer,antibodydetectionandstrippingmembranes.

·WesternBlots(WilliamH.Heidcamp)

• WesternBlot&Immunostaining(R&D)ProvidesveryniceandgeneralprotocolincludingWesternBlotProcedure,immunostainingProcedure(standardandrapid),helpfulhintsandtroubleshootingguide

·WesternBlottingProtocol(TheCellBIOLOGyandCytoskeletonGroup,HMS)

·ProteinGelandStaining(GottschlingLab)

·WesternBlotting(CorbettLab)

·ECMProtocolsWesternBlot(LTI)GeneralprotocolforECMassay.AmixtureofproteinisseparatedelectrophoreticallybySDS-PAGE,andtheindividualproteinbandsaretransferredtonitrocellulosepaper.Specificantibodyisthenusedtoprobeforanyofthebandsitmightbindto,andthenonboundantibodyiswashedaway.Theboundantibodyisthendetectedbytheadditionofasecondantibodyagainsttheimmunoglobulinspeciescontainedinthefirstantibody.Thissecondantibodyislabeledwithanenzyme,andthespecificproteinbandcanthenbevisualizedbytheadditionofanenzymesubstratecontainingacolordeveloper.

• WesternBlottingUsingChemiluminscence(PeterJ.HansenLab)
• WesternBlottingAnalysis(BDPharMingen)
• WesternBlottingwithHorserADIshPeroxidaseConjugates(TransductionLaboratories)DetailedprotocolofWesternBlotting,fromproteinisolation,quantification,electrophoresis,transferandmore...
• WesternBlottingwithAlkalinePhosphataseConjugates(TransductionLaboratories)DetailedprotocolofWesternBlotting,fromproteinisolation,quantification,electrophoresis,transferandmore...
• WesternBlottingwithMonoclonalAntibodies(TransductionLaboratories)DetailedprotocolofWesternBlotting,fromproteinisolation,quantification,electrophoresis,transferandmore...
• WesternBlottingwithRabbitPolyclonalAntibodies(TransductionLaboratories)DetailedprotocolofWesternBlotting,fromproteinisolation,quantification,electrophoresis,transferandmore...
• WesternBlottingwithBiotinylatedAntibodies(TransductionLaboratories)DetailedprotocolofWesternBlotting,fromproteinisolation,quantification,electrophoresis,transferandmore...

·AlkalinePhosphatase(AP)StainingofWesternBlots.(MolecularGeneticsRecLab)

• MAPKinaseGelShiftAssay(PMCI)
• Anti-PhosphotyrosineWesternBlotting(PMCI)

·Enzyme-AssistedImmunoelectroblotitng(IEBorWesternBlotting)(MolecularBiologyTechniquesManual)ThisprotocoldescribestheuseofhorizontalblottingofsimpleSDS-PAgels,andsubsequentdetectionofproteinsusingrabbitantiseraandalkalinephosphatase-conjugatedgoat-anti-rabbitIgG,detectedusingbromo-chloro-indolylphosphate(BCIP)andNitro-bluetetrazolium(NBT)salts.

·ProteinDot-/Slot-Blotting(Schleicher&Schuell)Dot-blotsofproteinsimplifyquantitativeassaysbydirectapplicationofthesampletoamembraneorfiltersheetwithoutpriorfractionation.Thetechniqueisusedforcarryingoutbindingassaysonmultiplesamples,andisvaluableforstudiesinwhichpurificationorotherprocessingofthemoleculemayalterbiologicalactivity.Thelatteradvantagemakesthedot-blottechniqueidealforpreliminaryscreeningtodeterminetheeffectofblottingorassayconditionsontheactivityofthesample.

·Alkalinephosphatase(AP)stainingofWesternblots(MolecularGeneticsNetworkLab)

·CDP-StarWesternBlotDetection(GimilaLab)

FarWesternBlotting

·FarWesternBlotting(KrauseLab)

SemiDryBlotting

·WesternBlottingUsingtheSemiPhorSemi-DryTransferUnit(GoldbergLab)Veryniceanddetailedprotocolwithillustration

·SemiDryBlotting

·WesternTransferUsingaSemidryApparatus(PMCI)

StrippingWesternMembranes

·StrippingandReprobingaWesternBlot(KPL)

·StrippingWesternMembranes(PaulBrennan)Reuseblotbystrippingantibodies

·StrippingWesternMembranes(MonicaColitti)Simpleprotocolforreusingblot

Troubleshootingandothers

·TroubleshootingWesternAndDot-BlotAssays(Schleicher&Schuell)

·Tipsandhintsforthestorageofantibodies(SynapticSystems)

·TooManyBandsonWesternBlot(SynapticSystems)Itisnotuncommonthat,contrarytothetheoreticalpredictions,severalbandsaredetected.AlthoughitispossIBLethattheantibodyisnotentirelyspecificfortheprotein,otherfactorsmayberesponsible

• WesternTroubleShooting(R&D)OffersgeneralguideforWesternblotting