X-GAL-solution-生命科学试剂-MCE

Hotline:400-820-3792Inhibitors•ScreeningLibraries•Proteinswww.MedChemEX-GAL(solution)Cat.No.:HY-DY2002CASNo.:7240-90-6分⼦式:C₁₄H₁₅BrClNO₆分⼦量:408.63作⽤靶点:FluorescentDye;Glycosidase作⽤通路:Others;MetabolicEnzyme/Protease储存⽅式:-20°C,protectfromlight.Avoidrepeatedfreezingandthawing*Insolvent:-80°C,6months;-20°C,1month(protectfrom

light.Avoidrepeatedfreezingandthawing)BIOLOGICALACTIVITY⽣物活性X-GAL(BCIG)(solution)⼀种⼴泛使⽤的显⾊β-半乳糖苷酶底物。在被β-半乳糖苷酶裂解之前，X-GAL是⼀种⽆⾊的化合物，裂解后X-GAL变成⼀种不溶性且可检测的蓝⾊化合物，这使得X-GAL可⽤于细克隆的蓝⽩筛选等技术中。X-GAL也可⽤于检测β-半乳糖苷酶的活性[1][2]。溶剂及浓度：DMF：20mg/mL体外研究1.Solutionpreparation[1]1.1StocksolutionStorage:Storeat-20°Cor-80°Cindarkafteraliquoting.Avoidrepeatedfreezingandthawing.1.2PreparationofworkingsolutionDiluteX-galwith1mLagarmedium,andadd1μLofIPTG(HY-15921)(24mg/mL)and1μLofAMP(HY-A0181)(100mg/mL)(optimizedaccordingtotheexperiment).Thecorrespondingstocksolutioncanbedilutedaccordingtotheactualsituation.NotethatifthesolventisDMSO,thecytotoxicityofDMSOmustbeconsidered,andasolventcontrolshouldbeprepared;ifthesolventispurewater,theworkingsolutionneedstobefilteredandsterilizedbeforeaddingcells.Note:Theworkingsolutionshouldbepreparedandusedimmediately.Keepitawayfromlight.2.Blue/whitecolonyscreening2.1AftertheDNAfragmentisinsertedintothepUCseriesvector(orothervectorswithlacZ,Ampgenes)andthentransformedintolacZ-deficientcells,applytheabove-mentionedX-gal,IPTG,andAMPplateculturemedium.2.2Incubateat37°Cuntilblue-whitecoloniesappearontheagarsurface.2.3Cultivatetheblue-whitecoloniesfurtherandidentifythembydoubleenzymedigestion.1/2MasterofBioactiveMolecules—您⾝边的抑制剂⼤师www.MedChemE2.4Aspiratethedyeworkingsolution,washwithPBS2-3times,andobserveunderamicroscope.Whenthecells(HUVECforexample)weregrownindishesto80%confluence,50μmol/LH2O2wereaddedinthemediumfor12h[2].Subsequently,cellswereharvestedwithtrypsin/EDTAandwashedthreetimeswithPBSandfurtherfixedwith4%formaldehydefor10min.Ascontrol,cellswithoutH2O2treatmentwerepreparedinthesameway.CellswerestainedwithbyX-galat37°C.Theintestinaltissuewasexcisedandcutlongitudinally.Aftertheintestinalcontentwasrinsedoffwithphosphatebuffersolution,theintestinaltissuewasplacedwiththeinnersurfacefacingupontheslide[2].Next,asinglebluecolonycontainingβ-gal(E.coliDH5αcontainingpUC18)wasdilutionin100μLPBSandthen10μLwereaddedinthetissue.Afterincubationfor1h,thetissueswerewashedwithPBSsolution.Ascontrol,tissueswithoutthebluecolonywerepreparedinthesameway.Alltissueswerefixedwith4%formaldehydefor10minandwerefurtherstainedbyaddingX-galat37°C.REFERENCES[1].Sanchez-RamosJ,etal.TheX-galcautioninneuraltransplantationstudies.CellTransplant.2000Sep-Oct;9(5):657-67.[2].LiS,etal.In-situSERSreadoutstrategytoimprovethereliabilityofbeta-galactosidaseactivityassaybasedonX-galstaininginshorteningincubationtimes.Talanta.2021Nov1;234:122689.McePdfHeightCaution:Producthasnotbeenfullyv