Enzolifesciences/Nitric oxide synthase (endothelial) polyclonal antibody/ALX210505R100/100µ

ProductSpecification:

AlternativeName:	eNOS,NOSIII

Host:	Rabbit

Immunogen:	Syntheticpeptidecorrespondingtoaa598-612(P⁵⁹⁸YNSSPRPEQHKSYK⁶¹²C)ofbovineendothelialnitricoxidesynthase(eNOS;NOSIII).

UniProtID:	P29473

Speciesreactivity:	Human,Mouse,Rat Dog

Crossreactivity:	Doesnotcross-reactwithinducibleNOS(iNOS;NOSII)orneuronalNOS(nNOS;NOSI).

Applications:	ICC,WB

RecommendedDilutions/Conditions:	Immunocytochemistry(1:250) WesternBlot(1:1,000) Suggesteddilutions/conditionsmaynotbeavailableforallapplications. Optimalconditionsmustbedeterminedindividuallyforeachapplication.

ApplicationNotes:	ImmunohistochemicalstainingofeNOS(NOSIII)inmousecardiactissuewithProd.No.ALX-210-505resultsinthestainingoftheendothelium. Detectsabandof~140kDabyWesternblot.

Formulation:	Liquid.NeatserumdilutedinPBScontaining0.05%sodiumazide.

Handling:	Avoidfreeze/thawcycles.

Shipping:	ShippedonBlueIce

LongTermStorage:	-20°C

Protocol:	ImmunocytochemistryofNOS Growcellstonearconfluenceonculture-wellslidesinRPMI1640mediumcontaining10%bovinecalfserum,2mMglutamineandpenicillin/streptomycin. ForstudyofiNOS,treatcellsfor16hourswithfreshmediumcontainingeither:noaddition(control),interferon-γ(50ng/ml),LPS(30µg/ml)orIFNandLPS. Washcells3x5minutesinPBS. Permeabilizewith2%saponinfor10minutesatroomtemperature. Fixin4%formalinfor10minutesatroomtemperature. Wash3x5minutesinPBS. Incubatefor20minutesatroomtemperaturewithantibodytoeNOS(Prod.No.ALX-210-505/1)dilutedwithPBSatsuggestedstartingdilution. Wash3x5minutesinPBS. Incubatewithbiotinylatedsecondaryantibodytorabbit,thenwithanavidin-biotincomplexconjugatedtoalkalinephosphatase. ReacttoeffectwithFastRed. WesternblotofNOS ForthestudyofiNOS,preparecytosolicextractsfromcellsthatarenottreatedanddonotexpressiNOSactivity(control)orcellsinducedtosynthesizeNObypretreatmentfor16hourswithacombinationpfLPS(30µg/ml)andinterferon-γ(50ng/ml). RuncellextractsonSDS-PAGEusinga4-12%TRIS-glycinegel.Transfertoblottingmembrane. Blockmembranein5%driedmilkinPBScontaining1%Tween-20(PBS-T). Wash3x5minutesinPBS. Incubatefor1houratroomtemperaturewithantibodytoNOSdilutedinPBS. Wash3x5minutesinPBS. Incubatefor1hourwithsecondaryantibodytorabbitcoupledtobiotin,thenwithstreptavidinconjugatedtoalkalinephosphatase. Wash3x5minutesinPBS. ReacttoeffectwithBCIPorLumi-Phos530 Note:Theprocedureslistedaboveareintendedonlyasaguide.Optimalconditionsmustbedeterminedindividuallyforeachapplication.

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Enzolifesciences/Nitric oxide synthase (endothelial) polyclonal antibody/ALX210505R100/100µ

ProductSpecification: