Type:MouseIgG
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
Immunogen:N-terminalregion,aminoacids1-608ofCas9sequenceCDJ55032.1fromStreptococcuspyogenes,expressedinandpurifiedfromE.coli.
ThediscoveryofCRISPR(ClusteredRegularlyInterspacedShortPalindromicRepeats)haschangedthefieldofgeneediting.TheserepeatedsequencesarefoundinbacterialgenomeswithshortDNAsequencesderivedfromviruseswhichhaveinfectedthebacteriainterspaced.ThesevirallyderivedsequencescanmakeshortRNAsequenceswhichcanhybridizewithspecificviralDNAandtargetanuclease,suchasCas9,totheviralsequence.So,ifthebacteriaareinfectedbythisvirusagain,Cas9canbedirectedtocleavethespecificviralsequenceandsoinactivatethevirus.BycarefuldesignoftheRNAsequencethesystemcanbeusedtospecificallycutDNAvirtuallyanywhere,includinginlivinghumanandothermammaliancells.Thisallowsinexpensivegeneeditingwithunprecedentedease,andmucheffortisgoingintorefiningtheCas9enzymesandtheirrelativesforuseinmammaliansystems.
Image:TransfectedHek293cellsoverexpressingtheN-terminal1-608aminoacidsof S.pyogenes Cas9.ThecellswerestainedwithMO22174inred,andthesecellsalsoappearyellowsincewecounterstainedwithourrabbitCas9antibody,RA22126tothesameconstructingreen,givingayellowcolor.TheN-terminalconstructcontainsanuclearlocalizationsequenceandsoispredominantlynuclearinlocalization.MostHek293cellsinthisfieldarenottransfectedsoonlythenucleiofthesecellscanbevisualizedwiththeblueDAPIDNAstain.
SeveralvarietiesofCas9havebeenstudiedandthereappeartobeseveralotherrelatedenzymeswithsimilarpropertiesinbacteria.TwoCas9homologsincludeStreptococcuspyogenseandStaphylococcusaureus.Staphylococcusaureus issignificantlysmallerandsopresentslessproblemswhenpackagedintovectors.The S.pyogenes proteinisratherlargeat1,368aminoacids,~160kDa,sothecorrespondingDNAisalsoratherlargeatabout4.2kb.Ourantibodyisamousemonoclonalraisedagainst aminoacids1-608ofCas9from S.pyogenes andbindstheimmunogentransfectedintocellsonwesternblotsandinimmunocytochemistry.Thehomologousregionofthe S.aureus Cas9isnotcloselyrelatedinaminoacidsequenceand,asexpected,thisantibodydoesnotrecognizethatprotein.
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