RabbitIgG(H+L)Cross-AdsorbedSecondaryAntibody(A-11008)inIF

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.

ProductDetails

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

ProductSpecificInformation

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.

Background/TargetInformation

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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