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
| TESTEDAPPLICATIONS | DILUTION |
|---|---|
| Immunocytochemistry(ICC) | 0.2µg/ml |
| Immunofluorescence(IF) | 1:2000 |
| Immunohistochemistry(IHC) | 1-10µg/ml |
| PUBLISHEDAPPLICATIONS | |
|---|---|
| Immunohistochemistry(IHC) | See6publicationsbelow |
| Immunocytochemistry(ICC) | See17publicationsbelow |
| Immunohistochemistry(Paraffin)(IHC(P)) | See3publicationsbelow |
| Immunohistochemistry(Frozen)(IHC(F)) | See5publicationsbelow |
| FlowCytometry(Flow) | See1publicationsbelow |
| MiscellaneousPubMed(MISC) | See36publicationsbelow |
| 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.
