MouseIgG(H+L)HighlyCross-AdsorbedSecondaryAntibody(A-21202)inIF

ImmunofluorescenceanalysisofDonkeyanti-MouseIgG(H+L)SecondaryAntibody,AlexaFluor488conjugate(Product#A-21202)wasperformedusingMCF-7cellsstainedwithCytokeratin19MouseMonoclonalAntibody(Product#MA5-12613).Thecellswerefixedwith4%paraformaldehydefor10minutes,permeABIlizedwith0.1%Triton™X-100for10minutes,blockedwith1%BSAfor1hourandlabeledwithmouseprimaryantibody(1:250dilution)for3hoursatroomtemperature.Donkeyanti-MouseIgG(H+L)SecondaryAntibody,AlexaFluor488conjugatewasusedatconcentrationof0.2µg/mlinphosphatebufferedsalinecontaining0.2%BSAfor45minutesatroomtemperature,fordetectionofcytokeratin19inthemembrane(Panela:green).Nuclei(Panelb:blue)werestainedwithDAPIinSlowFade®GoldAntifadeMountant(Product#S36938).F-actinwasstainedwithRhodaminePhalloidin(Product#R415,1:300)(Panelc:red).Paneldrepresentsthecompositeimage.Nononspecificstainingwasobservedwiththesecondaryantibodyalone(panelf),orwithanisotypecontrol(panele).Theimageswerecapturedat60X 

ProductDetails

Speciesreactivity	Mouse
Host/Isotype	Donkey/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_141607
Target	IgG
AntibodyForm	WholeAntibody

ProductSpecificInformation

Tominimizecross-reactivity,thesedonkeyanti-mouseIgGwholeantibodieshavebeenaffinity-purifiedandshowminimumcross-reactivitytobovine,chicken,goat,guineapig,hamster,horse,human,mouse,rat,andsheepserumproteins.Cross-adsorptionorpre-adsorptionisapurificationsteptoincreasespecificityoftheantibodyresultinginhighersensitivityandlessbackgroundstaining.Thesecondaryantibodysolutionispassedthroughacolumnmatrixcontainingimmobilizedserumproteinsfrompotentiallycross-reactivespecies.Onlythenonspecific-bindingsecondaryantibodiesarecapturedinthecolumn,andthehighlyspecificsecondariesflowthrough.Thebenefitsofthisextrastepareapparentinmultiplexing/multicolor-stainingexperiments(e.g.,flowcytometry)wherethereispotentialcross-reactivitywithotherprimaryantibodiesorintissue/cellfluorescentstainingexperimentswheretheremaybethepresenceofendogenousimmunoglobulins.

AlexaFluordyesareamongthemosttrustedfluorescentdyesavailabletoday.Invitrogen™AlexaFluor488dyeisabright,green-fluorescentdyewithexcitationideallysuitedtothe488nmlaserline.Forstablesignalgenerationinimagingandflowcytometry,AlexaFluor488dyeispH-insensitiveoverawidemolarrange.Probeswithhighfluorescencequantumyieldandhighphotostabilityallowdetectionoflow-abundanceBIOLOGicalstructureswithgreatsensitivity.AlexaFluor488dyemoleculescanbeattachedtoproteinsathighmolarratioswithoutsignificantself-quenching,enablingbrighterconjugatesandmoresensitivedetection.Thedegreeoflabelingforeachconjugateistypically2-8fluorophoremoleculesperIgGmolecule;theexactdegreeoflabelingisindicatedonthecertificateofanalysisforeachproductlot.

Usingconjugatesolutions:Centrifugetheproteinconjugatesolutionbrieflyinamicrocentrifugebeforeuse;addonlythesupernatanttotheexperiment.Thisstepwillhelpeliminateanyproteinaggregatesthatmayhaveformedduringstorage,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.

Oursecondaryantibodyconjugatesaremostcommonlypreparedbyimmunizingthehostanimalwithapooledpopulationofimmunoglobulinsfromthetargetspeciesandcanbefurtherpurifiedandmodified(e.g.,immunoaffinitychromatography,antibodyfragmentation,labelconjugation,etc.)togeneratehighlyspecificreagents.Inthefirstroundofpurification,wholeimmunoglobulinsbindingtotheimmunizingantibodyarerecoveredandmainlyconsistofthe~150-kDaIgGclass.Furtherpurification,forexample,withProteinAorG,removesallunwantedimmunoglobulinclassesexcepttheaffinity-purifiedantibodiesthatreactwiththetarget-specificimmunoglobulinheavyand/orlightchains.

ForResearchUseOnly.Notforuseindiagnosticprocedures.Notforresalewithoutexpressauthorization.

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