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ImmunofluorescenceanalysisofGoatanti-RabbitIgG(H+L)Cross-AdsorbedSecondaryAntibodyAlexaFluor?488conjugatewasperformedusingHeLacellsstainedwithalphaTubulinRabbitPolyclonalAntibody(Product#PA5-16891).Thecellswerefixedwith4%paraformaldehydefor10minutes,permeABIlizedwith0.1%Triton?X-100for10minutes,blockedwith1%BSAfor1hourandlabeledwith2μg/mlRabbitprimaryantibodyfor3hoursatroomtemperature.Goatanti-RabbitIgG(H+L)Cross-AdsorbedSecondaryAntibodyAlexaFluor?488conjugate(Product#A-11008)wasusedataconcentrationof4μg/mlinphosphatebufferedsalinecontaining0.2%BSAfor45minutesatroomtemperature,fordetectionofalphaTubulininthecytoplasm(Panela:green).Nuclei(Panelb:blue)werestainedwithDAPIinSlowFade?GoldAntifadeMountant(Product#S36938).F-actinwasstainedwithRhodaminePhalloidin(Product#R415,1:300)(Panelc:red).Paneldrepresentsthecompositeimage.Nononspecificstainingwasobservedwiththesecondaryantibodyalone(panelf),orwithanisotypecontrol(panele).Theimageswerecapturedat60Xmagnification.
| TESTEDAPPLICATIONS | DILUTION |
|---|---|
| FlowCytometry(Flow) | 1-10μg/mL |
| Immunocytochemistry(ICC) | 4μg/mL |
| Immunofluorescence(IF) | 4μg/mL |
| PUBLISHEDAPPLICATIONS | |
|---|---|
| Immunohistochemistry(Frozen)(IHC(F)) | See20publicationsbelow |
| Immunohistochemistry(IHC) | See20publicationsbelow |
| Immunocytochemistry(ICC) | See26publicationsbelow |
| Immunohistochemistry(Paraffin)(IHC(P)) | See8publicationsbelow |
| FlowCytometry(Flow) | See2publicationsbelow |
| WesternBlot(WB) | See3publicationsbelow |
| Immunohistochemistry-FreeFloating(IHC(Free)) | See1publicationsbelow |
| ChIPassay(ChIP) | See1publicationsbelow |
| MiscellaneousPubMed(MISC) | See472publicationsbelow |
| Speciesreactivity | Rabbit |
| Host/Isotype | Goat/IgG |
| Class | Polyclonal |
| Type | SecondaryAntibody |
| Immunogen | GammaImmunoglobinsHeavyandLightchains |
| Conjugate | AlexaFluor?488 |
| Excitation/EmissionProfile | Viewspectra  |
| Form | liquid |
| Concentration | 2mg/ml |
| Purification | purified |
| Storagebuffer | PBS,pH7.5 |
| Contains | 5mMsodiumazide |
| Storageconditions | 4°C,storeindark |
| RRID | AB_143165 |
| Target | IgG |
| CrossAdsorption | AgainsthumanIgG,humanserum,mouseIgG,mouseserumandbovineserum |
| AntibodyForm | WholeAntibody |
AlexaFluordyesareamongthemosttrustedfluorescentdyesavailabletoday.Invitrogen?AlexaFluor?488dyeisabright,green-fluorescentdyewithexcitationideallysuitedtothe488nmlaserline.Forstablesignalgenerationinimagingandflowcytometry,AlexaFluor488dyeispH-insensitiveoverawidemolarrange.Probeswithhighfluorescencequantumyieldandhighphotostabilityallowdetectionoflow-abundanceBIOLOGicalstructureswithgreatsensitivity.AlexaFluor488dyemoleculescanbeattachedtoproteinsathighmolarratioswithoutsignificantself-quenching,enablingbrighterconjugatesandmoresensitivedetection.Thedegreeoflabelingforeachconjugateistypically2-8fluorophoremoleculesperIgGmolecule;theexactdegreeoflabelingisindicatedonthecertificateofanalysisforeachproductlot.
Thegoatanti-rabbitIgGwholeantibodyconjugatesaremostcommonlypreparedbyimmunizingthehostanimalwithapooledpopulationofimmunoglobulinsfromthetargetspeciesandcanbefurtherpurifiedandmodified(e.g.,immunoaffinitychromatography,antibodyfragmentation,labelconjugation,etc.)togeneratehighlyspecificreagents.Inthefirstroundofpurification,wholeimmunoglobulinsbindingtotheimmunizingantibodyarerecoveredandmainlyconsistofthe~150-kDaIgGclass.FurtherpurificationwithProteinAorGremovesallimmunoglobulinclassesexceptIgGsuchthattheaffinity-purifiedantibodiesreactwithIgGheavychainsandallclassesofimmunoglobulinlightchainsfromrabbit.Tominimizecross-reactivity,thesegoatanti-rabbitwholeantibodieshavebeencross-adsorbedagainsthumanIgG,humanserum,mouseIgG,mouseserum,andbovineserum.Cross-adsorptionorpre-adsorptionisapurificationsteptoincreasespecificityoftheantibodyresultinginhighersensitivityandlessbackgroundstaining.Thesecondaryantibodysolutionispassedthroughacolumnmatrixcontainingimmobilizedserumproteinsfrompotentiallycross-reactivespecies.Onlythenonspecific-bindingsecondaryantibodiesarecapturedinthecolumn,andthehighlyspecificsecondariesflowthrough.Thebenefitsofthisextrastepareapparentinmultiplexing/multicolor-stainingexperimentswherethereispotentialcross-reactivitywithotherprimaryantibodiesorinimmunohistochemistryexperimentswheretherearemaybethepresenceofendogenousimmunoglobulins.Forahighlycross-adsorbedsecondaryantibodyequivalent(orequivalentsecondaryantibodypreparation),pleaseseeproductcatalognumber:A11034.
Usingconjugatesolutions:Centrifugetheproteinconjugatesolutionbrieflyinamicrocentrifugebeforeuse;addonlythesupernatanttotheexperiment.Thisstepwilleliminateanyproteinaggregatesthatmayhaveformedduringstorage,therebyreducingnonspecificbackgroundstaining.Becausestainingprotocolsvarywithapplication,theappropriatedilutionofantibodyshouldbedeterminedempirically.Forthefluorophore-labeledantibodiesafinalconcentrationof1-10μg/mLshouldbesatisfactoryformostimmunohistochemistryandflowcytometryapplications.
WeofferanextensivelineofInvitrogen?secondaryantibodyconjugateswithwell-characterizedspecificityandlabeledwithawideselectionofpremiumfluorescentdyes,includingInvitrogen?AlexaFluor?fluorescentdyes.Fluorescentsecondaryantibodyconjugatesareusefulinthedetection,sorting,orpurificationofitsspecifiedtargetandidealforfluorescencemicroscopyandconfocallaserscanningmicroscopy,flowcytometry,andfluorescentwesterndetection.ThebreadthoffluorescentMarkersweofferallowsourreagentstobetailoredtoalmostanyfluorescentdetectionsystem.
Secondaryantibodiesmaybeprovidedinthreeformats:wholeIgG,divalentF(ab')2fragments,andmonovalentFabfragments.Becauseofthehighdegreeofconservationinthestructureofmanyimmunoglobulindomains,mostclass-specificsecondaryantibodiesmustbeaffinity-purifiedandcross-adsorbedtoachieveminimalcross-reactionwithotherimmunoglobulins.
ForResearchUseOnly.Notforuseindiagnosticprocedures.Notforresalewithoutexpressauthorization.
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