Type:RabbitIgG
Applications:ICC;IF;WB
E=ELISA;FACS;FC=FlowCytometry;FPLC=FastProteinLiquidChromatography;GF=GravityFlow;HPLC=HighPerformanceLiquidChromatography;ICC=Immunocytochemistry;IF=Immunofluorescence;IHC=Immunohistochemistry;IP=Immunoprecipitation;NAC=Non-adherentCellAssays;NB=NeutralizationofBioactivity;SE=SandwichELISA;TPE=TargetedProteinExpression;WB=Westernblotting;;AC=AdherentCellAssays;FM=FluorescentMicsroscopy;;;BSC-CM5=BiacoreSensorChipCM5;BSM=BiosactiveSmallMoleculeorPeptide;CDM=CellDifferentiationMedia;;;;;;HealthandFitness;;;DNAExtraction/Purification;;InvivoLikeAssaysSpeciesReactivity:NotApplicable
B=Bovine;Ca=Cat;Ch=Chicken;D=Dog;EQ=Equine;GP=GuineaPig;H=Human;M=Mouse;P=Porcine;Pr=Primate;R=Rat;Rb=Rabbit;Y=Yeast;Xe=Xenopus;Ze=Zebrafish;;;;NA-NotApplicable;STP=Step-TactinProteins;AllFormat:AffinityPurified-liquid
ThediscoveryofCRISPR(ClusteredRegularlyInterspacedShortPalindromicRepeats)haschangedthefieldofgeneediting.TheserepeatedsequencesarefoundinbacterialgenomeswithshortDNAsequencesderivedfromviruseswhichhaveinfectedthebacteriainterspaced.ThesevirallyderivedsequencescanmakeshortRNAsequenceswhichcanhybridizewithspecificviralDNAandtargetanuclease,suchasCas9,totheviralsequence.So,ifthebacteriaareinfectedbythisvirusagain,Cas9canbedirectedtocleavethespecificviralsequenceandsoinactivatethevirus.BycarefuldesignoftheRNAsequencethesystemcanbeusedtospecificallycutDNAvirtuallyanywhere,includinginlivinghumanandothermammaliancells.Thisallowsinexpensivegeneeditingwithunprecedentedease,andmucheffortisgoingintorefiningtheCas9enzymesandtheirrelativesforuseinmammaliansystems.
Image:Hek293cellculturetransfectedwithaconstructincludingtheCterminal814-1372aminoacidsofStaphylococcuspyogenesCas9.ThecellswerestainedwithRA22126inredandalsowithachickenantibodytoS.pyogenesCas9ingreen.AcellexpressingCas9thereforeappearsyellow.MostHek293cellsarenottransfectedsoonlythenucleusofthesecellscanbevisualizedwiththeblueDNAstain.
SeveralvarietiesofCas9havebeenstudiedandthereappeartobeseveralotherrelatedenzymeswithsimilarpropertiesinbacteria.TwoCas9homologsincludeStreptococcuspyogenseandStaphylococcusaureus.Staphylococcusaureusissignificantlysmallerandsopresentslessproblemswhenpackagedintovectors.TheS.pyogenesproteinisratherlargeat1,368aminoacids,~160kDa,sothecorrespondingDNAisalsoratherlargeatabout4.2kb.Ourantibodyisanaffinitypurifiedpolyclonalraisedinrabbitagainstamixtureoftworecombinantconstructscorrespondingtoaminoacids1-608and814-1372ofCas9fromtheStreptococcuspyogenesandbindsboththeseproteinstransfectedintocellsandonwesternblots,andalsorecognizesthefulllengthprotein.ThehomologousregionoftheS.aureusCas9isnotcloselyrelatedinaminoacidsequenceand,asexpected,thisantibodydoesnotrecognizethatprotein.
MoreLinks:
GeneTools Cas9fromStaphylococcusaureus-RabbitWholeSerumAntibody | Cas9fromSteptococcuspyogenese-MouseMonoclonalAntibody Cas9fromStaphylococcusaureus-ChickenIgYPrepAntibody | Cas9fromStaphylococcusaureus- MouseMonoclonalAntibody |
![Picture[2]](images/neuromics/2017/A8x12145x66x1.jpg)
Image:WesternblotanalysisofRA22126
Blotof100ng(lane1)and10ng(lane2)offulllengthCas9proteinfromStreptococcuspyogeneswasprobedwithRA22126at1:2,000dilution.ThisantibodyrecognizesfulllengthCas9proteinat160kDaandaproteolyticfragmentat130kDa.
