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蚂蚁淘在线 / 品牌中心 / Lucigen / Lucigen/Expresso® CMV Cloning & Expression System/49031-2/10 rxns

Lucigen/Expresso® CMV Cloning & Expression System/49031-2/10 rxns

价格
￥7320.00
货号：49031-2
浏览量：127
品牌：Lucigen
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商品描述
  • Fast:5-second,directionalcloningwithnoenzymeincubations,ligation,orvectorprep.Watchvideo»
  • Precise:"Noscar"vectorresultsinhighlyaccuratesequencesandcorrectproteins.
  • SmallVector:3.4-kbvectorgiveshightransfectionefficiency&easydownstreammanipulation.
  • Robust:CMVpromoterprovidesstrong,constitutiveeukaryoticexpression.

TheExpresso®CMVCloningandExpressionSystemcombinesinstant,enzyme-free,directionalcloningwitha“noscar”vectorforeukaryoticgeneexpression.ThiscloningsystemisbasedonExpressioneering™Technology,whichusesinvivohomologousrecombinationtoseamlesslyclonePCR-amplifiedDNAintospeciallydesignedexpressionvectorswithoutpurificationorenzymaticincubations.It’sthefastest,easiestmammalianexpressionsystemavailable.

Expresso Vectors and Technology for Protein Expression
Figure1.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCR-amplifiedDNAintothepME-HAvector.Targetgenesareamplifiedwithprimersthatinclude18basesofoverlapwiththeendsoftheExpressovector.TheunpurifiedPCRampliconissimplymixedwiththepME-HAexpressionplasmidandimmediatelytransformedintothehighlycompetentcellsprovided.NoDNApurification,enzymetreatment,vectorpreparation,orligationstepsarerequired.
 

pME-HAVector

TheExpressoCMVCloningandExpressionSystemfeaturesthepME-HAexpressionvector,whichcontainsalltheelementsyouneed,andnothingyoudon't.TheCMVpromoterenablesstrong,constitutiveexpressioninthesmallestmammalianexpressionvectoravailable.The3.4kbvectorsupportshighertranfectionefficiencyandeasierdownstreammanipulation.CombinedwithExpressioneeringtechnologyforinstant,scar-freecloning,theresultissurprisinglysimplemammalianexpression.

Expresso Vectors and Technology for Protein ExpressionFigure2.pME-HAexpressionvectorTheCMVpromoterdrivesexpressionofthePCRinsert;ampandSV40promotersexpressthegeneforkanamycin/neomycinresistanceinbacteriaandmammaliancells.ThepUCorigingiveshighplasmidyields.CloneSmart®transcriptionterminators(T)preventtranscriptionintooroutofthevectorbackbone,increasingclonestABIlity.TheHAaffinitytagcanbefusedtothecarboxyterminusoftheexpressedprotein,ifdesired.

StrongExpression

ThepME-HAvectorprovidesrobustproteinexpression,greaterthanorequaltothatofthecommonly-usedvectorpCDNA3.1.ProteinexpressionfromtheGFPgene(0.8kb)andthefulllengthβ-galactosidasegene(3kb)wasdetectedbyWesternblottingagainsttheHAtaginCHO-K1andCOS-7transfectants(Figure3).β-galactosidaseexpressionwasalsomeasuredquantitativelyintransfectedCHO-K1cells(Figure4).

Expresso Vectors and Technology for Protein Expression
Figure3.ComparisonofproteinexpressionfrompME-HAandpcDNA3.1vectorsinCHOandCOScells.CHOandCOScellsweretransfectedwithconstructsencodingGreenFluorescentProtein(GFP)andbeta-galactosidase(β-gal)usingLipofectamine2000(Invitrogen)followingthemanufacturer'srecommendation.At48hposttransfection,wholecelllysateswerefractionatedbySDSPAGE,transferredtoaPVDFmembrane,anddetectedwithanti-HAantibody.ExpressionfromthepME-HAvectorwasequaltoorgreaterthanthatfromthe“standard”pcDNA3.1vector.
 
Multiplication of bGal
Figure4.CHO-K1cellsweretransfectedwithpME-HA-Bgal plasmidataratioof2:1(µlTransIT-2020transfectionreagent:µgDNA).CellswereassayedforBgalactivity24-hourspost-transfectionusingtheMammalianB-GalactosidaseAssayKit(ThermoScientific,#75707),andreadat405nmonaplatereader.Absorbancevaluesweredividedbybackgroundabsorbance(mock-transfectedcells).

ExpressionofDifficultProteins

TheuseofExpressioneeringtechnologyresultsinprecisecloning,withouttheadditionofunwantedsequence(suchasrestrictionsites)typicallypresentinmultiplecloningsites.TheminimalsizeofthepME-HAvectoralsocontributestoenhancedcloningcapability.NumerousGPCRgeneshavebeenclonedandexpressedinmammaliancellsusingtheExpressoCMVsystem.WesternblottingwasusedtodetectexpressionoftheHA-taggedGPCRs(Figure5),andfunctionalitywasconfirmedbyrADIoactiveligandbindingassays(Figure6).

Expresso Vectors and Technology for Protein Expression
Figure5.ExpressionofGPCRsfromthepME-HAvector.COS-7cellsweretransfectedwithconstructsencodingturkeybeta1-adrenergicreceptor(β1-AR)andhumanadenosineA1receptor(A1A)withTransIT-2020(MirusBioLLC)orLipofectamine2000(Invitrogen)followingmanufacturer'srecommendation.At48hourspost-transfection,wholecelllysateswerefractionatedbySDS-PAGE,transferredontoPVDFmembrane,anddetectedwithanti-HAantibody.

 

Single Point Assays
Figure6.RadioligandbindingdataforGPCRsexpressedfromthepME-HAvector.Cellsexpressingb1-ARwereanalyzedforradioligandbindingusing20nM[3H]-Dihydroalprenolol(Perkinelmer).Non-specificbindingwasdeterminedinpresenceofexcessunlabeledcompetitor,10μM(S)-(-)-Propanololhydrochloride.ReceptorboundligandwascapturedonGF/Bfiltersandcounted.CellsexpressingA1Areceptorwereanalyzedbybindingof10μM[3H]-DPCPX(PerkinElmer).Non-specificbindingwasdeterminedinpresenceofexcessunlabeledcompetitor,10μM8-Cyclopentyl-1,3-dipropylxanthine.BoundligandwascapturedonGF/Bfiltersandcounted.

ORDERINFORMATION

TheExpressoCMVCloning&ExpressionSystemcontainspre-processedpME-HAVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andrecoverymedium.Alsoincludedareβ-galactosidasepositivecontrolinsertDNA,transformationpositivecontrolpUCDNA,andforwardandreversePCRprimerstoconfirmclones. Lucigen基因组编辑和工程面临许多挑战。为您的编辑实验获取可靠的试剂不应该是其中之一。无论您是在突破CRISPR技术的界限，在体内产生基因敲除或敲入还是在进行序列消化或鉴定的体外反应，您都需要可靠的工具和酶。依靠CRISPRcraft™获得质量，可靠性和稳定的性能。