MouseIgG(H+L)Cross-AdsorbedSecondaryAntibody(A-11005)inIF

HumaniPSCStainingHumaniPSCswereculturedonglassslidesunderfeeder-freeconditionsinStemPro®hESCMedium(Product#A1000701).CellswerefixedandpermedwiththeImage-iT®Fixation/PermeABIlizationKit(Product#R37602).Oct4(green)expressionwasvisualizedusinganti-Oct4primaryAbandAlexaFluor®488secondaryAb(Product#A-11034).Tubulin(red)expressionwasvisualizedusinganti-tubulinprimaryAb(Product#32-2600)andAlexaFluor®594secondaryAb(Product#A-11005).Nuclei(blue)werelabeledwithNucBlue™FixedCellStain(Product#R37606).ImageswerecollectedontheFLoid™CellImagingStation(Product#4471136).

ProductDetails

Speciesreactivity	Mouse
Host/Isotype	Goat/IgG
Class	Polyclonal
Type	SecondaryAntibody
Immunogen	GammaImmunoglobinsHeavyandLightchains
Conjugate	AlexaFluor®594
Excitation/EmissionProfile	Viewspectra
Form	Liquid
Concentration	2mg/ml
Purification	purified
Storagebuffer	PBS,pH7.5
Contains	5mMsodiumazide
Storageconditions	4°C,storeindark
RRID	AB_2534073
Target	IgG
CrossAdsorption	AgainsthumanIgGandhumanserumpriortoconjugation
AntibodyForm	WholeAntibody

ProductSpecificInformation

Tominimizecross-reactivity,thesegoatanti-mouseIgGwholeantibodieshavebeencross-adsorbedagainsthumanIgGandhumanserumpriortoconjugation.Cross-adsorptionorpre-adsorptionisapurificationsteptoincreasespecificityoftheantibodyresultinginhighersensitivityandlessbackgroundstaining.Thesecondaryantibodysolutionispassedthroughacolumnmatrixcontainingimmobilizedserumproteinsfrompotentiallycross-reactivespecies.Onlythenonspecific-bindingsecondaryantibodiesarecapturedinthecolumn,andthehighlyspecificsecondariesflowthrough.Thebenefitsofthisextrastepareapparentinmultiplexing/multicolor-stainingexperiments(e.g.,flowcytometry)wherethereispotentialcross-reactivitywithotherprimaryantibodiesorintissue/cellfluorescentstainingexperimentswheretherearemaybethepresenceofendogenousimmunoglobulins.

AlexaFluordyesareamongthemosttrustedfluorescentdyesavailabletoday.Invitrogen™AlexaFluor594dyeisabright,red-fluorescentdyewithexcitationideallysuitedtothe594nmlaserline.Forstablesignalgenerationinimagingandflowcytometry,AlexaFluor594dyeispH-insensitiveoverawidemolarrange.Probeswithhighfluorescencequantumyieldandhighphotostabilityallowdetectionoflow-abundanceBIOLOGicalstructureswithgreatsensitivity.AlexaFluor594dyemoleculescanbeattachedtoproteinsathighmolarratioswithoutsignificantself-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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