| InputType: | NuclearExtracts,PurifiedEnzyme |
| ResearchArea: | DNAMethylation |
| TargetApplication: | ActivityMeasurement |
| VesselFormat: | 96-WellPlate |
| 100%Guarantee: | 6months |
TheEpigenase™ 5mC-HydroxylaseTETActivity/InhibitionAssayKit(Colorimetric)isacompletesetofoptimizedbuffersandreagentsformeasuringactivity/inhibitionoftotalcytosineoxygenaseTETenzymesinnuclearextractsorpurifiedTETisoforms(TETs1-3)fromabroadrangeofspeciessuchasmammals,plants,fungi,andbacteria,andinavarietyofformsincluding,butnotlimitedtoculturedcellsandfreshandfrozentissues. Thekithasthefollowingadvantagesandfeatures:
- 5hourcolorimetricassayprocedurewitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin5hours.
- DirectlymeasuresTEThydroxylaseactivityviaastraightforwarddetectionofTET-convertedhydroxymethylatedproducts.
- Innovativekitcompositionenablesbackgroundsignalstobeextremelylowandallowstheassaytobesimple,accurate,reliable,andconsistent.
- Eithercell/tissueextractsorpurifiedTETproteinscanbeused,whichallowsfordetectionofinhibitoryeffectsofTEThydroxylaseinhibitors invivo and invitro.
- Novelassayprincipleallowshighsensitivitytobeachieved.Theactivitycanbedetectedfromaslowas20ngofpurifiedTET1hydroxylase.
- Ahydroxymethylatedstandardisincluded,whichallowsthespecificactivityofTEThydroxylasestobequantified.
- StripmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis(96assays).
BackgroundInformation
5-hydroxymethylcytosine,asasixthDNAbasewithfunctionsintranscriptionregulation,hasbeendetectedtobeabundantinthehumanandmousebrainandembryonicstem(ES)cells.Inmammals,itcanbegeneratedbyoxidationof5-methylcytosine,areactionmediatedbytheten-eleventranslocation(TET)familyofcytosineoxygenases.
TheTETfamilyofcytosineoxygenasesincludesTET1,TET2,andTET3.TheseTETproteinsmaypromoteDNAdemethylationbybindingtoCpG-richregionstopreventunwanted DNAmethyltransferase activity,andbyconverting5mCto5hmCandfurtherto5caC(5-carboxylcytosine)throughhydroxylaseactivity.Itwasshownthatgenomic5hmClevelscorrelatewithTEThydroxylaseactivity.Inaddition, TET1wasshowntohavedualfunctionsintranscriptionactivationandrepressionbybindingdifferenttargetgenesinEScells.TET1isalsoafusionpartneroftheMLLgeneinacutemyeloidleukemiaandisconsideredanoncoprotein.TET2isfoundtobefrequentlymutatedinleukemiaandconsideredtoactastumorsuppressor.TET3wasdemonstratedtoplayauniqueroleinDNAmethylationreprogrammingprocessesinthemammalianzygote.Thus,activatingtumorsuppressorTETenzymessuchasTET2orinhibitingoncoproteinTETenzymessuchasTET1wouldbeimportantinbenefitingcancerdiagnositcsanddevelopingnewtarget-basedcancertherapeutics.
Principle&Procedure
Inthisassay,amethylatedsubstrateisstablycoatedontothemicroplatewells.ActiveTETs(notincluded)bindtothesubstrateandconvertthemethylatedsubstratetohydroxymethylatedproducts.TheTET-convertedhydroxymethylatedproductscanberecognizedwithaspecificantibody.Theratiooramountofhydroxymethylatedproducts,whichisproportionaltoenzymeactivity,canthenbecolorimetricallymeasuredbyreADIngtheabsorbanceinamicroplatespectrophotometeratawavelengthof450nm.TheactivityoftheTETenzymeisinturnproportionaltotheopticaldensityintensitymeasured.
WB(10XWashBuffer)
TAB(TETAssayBuffer)
TS(10XTETSubstrate)*
BS(BindingSolution)
HC5(TETAssayStandard,20µg/ml)*
HC6(CaptureAntibody,1000µg/ml*
HC7(DetectionAntibody,400µg/ml)*
HC8(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
Co-factor1*
Co-factor2*
Co-factor3*
8-WellAssayStrips(WithFrame)
*Formaximumrecoveryoftheproducts,centrifugetheoriginalvialpriortoopeningthecap.
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常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
有了源数据之后把源数据按照大小排列，
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
:)
我在做一细菌不同酸碱度生长状况时,发现这些奇怪现象:pH=3的培养基灭菌(TSB液体培养基)灭菌后pH上升到到9.2!而原来pH=9.0的降到8.7(基本没多少变化),请问各位大侠,这是什么原因?
一般做不同酸碱度生长实验时,该如何才能防止pH在湿热灭菌后基本不变化?
是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
