productinformationBackgroundHistone3(H3)locatedinnuclei,incorporatedintochromatin.PresentinnucleosometogetherwithH2A,H2BandH4.ImmunogenKLH-conjugatedsyntheticpeptidederivedfromknownH3sequences,inludingArABIdopsisthalianaH3.3P59169(At4g40030,At4g40040,At5g10980),H3.2P59226(At1g09200,At3g27360,At5g10390,At5g10400,At5g65360),H3-like2Q9FXI7(At1g19890)HostRabbitClonalityPolyclonalClonePuritySerumFormatLyophilizedQuantity2x50µlReconstitutionForreconstitutionadd50µlofsterilewatertoeachtubeStoragestorelyophilized/reconstitutedat-20°C;oncereconstitutedmakealiquotstoavoidrepeatedfreeze-thawcycles.Please,remembertospintubesbrieflypriortoopeningthemtoavoidanylossesthatmightoccurfromlyophilizedmaterialadheringtothecaporsidesofthetubes.Testedapplicationswesternblot(WB),immunocytochemistry(ICC)RelatedproductsAS111804 |RNApolymeraseIIsubunitB1,rabbitantibodyAS10710PRE|H3|histoneH3,pre-immuneserumcollectionofantibodiestoDNA/RNA/cellcyclePlantandalgalproteinextractionbufferSecondaryantibodiesAdditionalinformationcellular[compartmentMarker]ofnucleoplasm,loADIngcontrolantibodyforChlamydomonasreinhardtiiapplicationinformationRecommendeddilution1:5000withstandardECL(WB),1:100-1:500(ICC)Expected|apparentMW15|17kDaConfirmedreactivityArabidopsisthaliana,Brassicaoleracea,Capsicumannuum,Chlamydomonasacidophila,Chlamydomonasreinhardtii,Physcomitrellapatens,SaLicorniaeuropaea,Solanumlycopersicum,Solanumsogarandinum,Solanumtuberosum,Viciafaba,ZeamaysPredictedreactivitydicotsincluding:Vitisvinifera,monocots:Hordeumvulgare,Oryzasativa,Zeamays.Nicotianatabacum,Medicagosativa,Triticumaestivum,Pisumsativum,Brassicanapus. trees:Pinuspinaster,algae:Volvoxsp.NotreactiveinnoconfirmedexceptionsfrompredictedreactivityknowninthemomentAdditionalinformationProtocolforisolationofcytosolicandnuclearfractionscanbefoundhere.SpecificfluorescenceinICChasbeenobservedforinterphasenucleiaswellasaroundcentromerregion(whereSer10ofhistoneH3isphosphorylated)inmitoticchromosomes.SelectedreferencesSpijkermanetal.(2014).CO2acquisitioninChlamydomonasacidophilaisinfluencedmainlybyCO2,notphosphorus,availability.PhotosynthRes.2014Sep;121(2-3):213-21.doi:10.1007/s11120-014-0016-6.Epub2014Jun7.Rihanetal.(2014).TheeffectofmolyBDenumonthemolecularcontrolofcoldtoleranceincauliflower(Brassicaoleraceavar.botrytis)artificialseeds.PlantCell,TissueandOrganCulture(PCTOC)April2014Szabalaetal.(2014).AccumulationofacidicSK3dehydrinsinphloemcellsofcold-anddrought-stressedplantsoftheSolanaceae.Planta,Jan7.WobbeandNixon(2013).ThemTERFproteinMOC1terminatesmitochondrialDNAtranscriptionintheunicellulargreenalgaChlamydomonasreinhardtii.NucleicAcidsRes.May6.applicationexample 1.2μgofArabidopsisthalianachromatin-enrichedfraction(1)and3.75µgoftotalproteinfrom4-weeks-oldArabidopsisthalianaleaves(2),andwereseparatedon12%SDS-PAGEandblotted50minstoImmobilon-P(Millipore,semi-dry)PVDFmembrane.BlotswereblockedimmediatelyfollowingtransferinMTBS-T(5%milk)for30minsatroomtemperaturewithagitation.Blotswereincubatedintheprimaryantibodyatadilutionof1:5000for1hatroomtemperaturewithagitation.Theantibodysolutionwasdecantedandtheblotwasrinsedbrieflytwice,thenwashed3timesfor3mininTBS-Tatroomtemperaturewithagitation.Blotswereincubatedinsecondaryantibody(anti-IgGhorseradishperoxidaseconjugated,fromAgrisera,AS09602)dilutedto1:20000for30minsatroomtemperaturewithagitation.Theblotswerewashedasaboveanddevelopedfor5minwithECLdetectionreagent(Roche)accordingtothemanufacturersinstructions.Exposuretimewas30seconds.Doublebandinchromatine-enrichedfraction(1)hasbeenoutcompetedinpeptideneutralizationassaybypeptideusedtoelicitH3antibodies.Chromatinizolationwascarriedoutasdescribed(Zilbermanetal.2008)withminormodifications.CourtesyofWeronikaSuraandDr.PiotrA.Ziolkowski(DepartmentofBiotechnology,AdamMickiewiczUniversity,Poznan,Poland)  30μgofChlamydomonasreinhardtiitotal(“Cells”)ornuclei-enriched(“Nuclei”)proteinswereseparatedona13%SDS-PAGEgelandblotted90minutestoAmershamTMHybondTM-ECL(poresize0.2μm).MembraneswereblockedinTBST5%milkforonehouratroomtemperatureandincubatedat4°CovernightinTBST5%milkwiththeAgriseraprimaryH3antibodyatadilutionof1:5000.AfterthreewashesinTBST,blotswereincubatedonehouratroomtemperaturewithAgriseraHRP-conjugatedGoatanti-rabbitIgG(H&L)diluted1:50000inTBST5%milk.SignalwasvisualizedwithstandardECLonAmershamHyperfilmTMECLafter1minuteexposure.MolecularweightisdeterminedbytheBroadRangeProteinMolecularWeightMarker(Promega).CourtesyofDr.LeonardoMagneschi,Hippler’sLab,InstitutfürBIOLOGieundBiotechnologiederPflanzen(IBBP),WestfälischeWilhelms-UniversitätMünster,Germany Immunocytochemicalassayswereperformedaccordingtothemethoddescribedearlier(RybaczekandMaszewski2006).ExcisedapicalpartsofViciafabaroots(1.5mmlong)werefixedfor45min(18°C)inPBS-buffered3.7%paraformaldehyde,washedseveraltimeswithPBSandplacedinacitricacid-buffereddigestionsolution(pH5.0;37°Cfor45min)containing2.5%pectinase(Fluka),2.5%cellulase(OnozukaR-10;Serva)and2.5%pectoliase(ICN).Afterremovingthedigestionsolution,roottipswerewashed3timesinPBS,rinsedwithdistilledwaterandsquashedontoSuperFrostPlusglassslides(Menzel-Gläser).Air-driedslideswerepretreatedwithPBS-buffered5%BSAat20°Cfor50minandincubatedovernightinahumidifiedatmosphere(4°C)withrabbitantibodyraisedagainstH3histone(Agrisera),dissolvedinPBScontaining1%BSA(atadilutionof1:50).Followingincubation,slideswerewashed3timeswithPBSandincubatedfor1h(18°C)withAgriserasecondarygoatanti-rabbitIgGDyLight®488antibody(AS09633,1:1000).NuclearDNAwasstainedwith4’,6-diamidino-2-phenyl-indole(DAPI,0.4μg/ml;Sigma-Aldrich).FollowingwashingwithPBS,slideswereairdriedandembeddedinVectashieldMountingMediaforFluorescence(VectorLaboratories).ObservationsweremadeusingOptiphot-2fluorescencemicroscope(Nikon)equippedwithB-2Afilter(bluelight;λ≈495nm)forDyLight-conjugatedantibodiesandUV-2Afilter(UVlight;λ≈365nm)forDAPI.AllimageswererecordedatexactlythesametimeofintegrationusingDXM1200CCDcamera.CourtesyDr.DorotaRybaczek,LodzUniversity,Poland