| InputType: | NuclearExtractsorPurifiedEnzymes |
| ResearchArea: | RNAMethylation |
| TargetApplication: | AmountQuantitation |
| VesselFormat: | 96-WellPlate |
| 100%Guarantee: | 6months |
The Epigenase™m6ADemethylaseActivity/InhibitionAssayKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallymeasuretheactivity/inhibitionoftotalm6Ademethylasesusingnuclearextractsorpurifiedm6AdemethylaseslikeFTOandALKBH5fromabroadrangeofspeciessuchasmammalian,plant,fungal,andbacterial,inavarietyofformsincluding,butnotlimitedto,culturedcellsand,freshandfrozentissues.Thiskithasthefollowingadvantagesandfeatures:
- Colorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin5hours.
- Innovativekitcompositionenablesbackgroundsignalstobeextremelylowandallowstheassaytobesimple,accurate,reliable,andconsistent.
- Bothcell/tissuenuclearextractsandpurifiedproteinscanbeused,whichallowsdetectionofinhibitoryeffectsofm6Ademethylaseinhibitorinvivoandinvitro.
- Novelassayprincipleallowshighsensitivitytobeachieved.Theactivitycanbedetectedfromaslowas2µgofnuclearextractsor50ngofpurifiedenzymes.
- Theassaystandardisincluded,whichallowsthespecificactivityofm6Ademethylasetobequantified.
- Strip-wellmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis.
BackgroundInformation
N6-methyladenosine(m6A)isthemostcommonandabundantmodificationonRNAmoleculespresentineukaryotes.Recently,DNAm6AisalsoidentifiedinmulticellulareukaryotesincludingCaenorhaBDitiselegansandDrosophilamelanogaster,andfurThermoreidentifiedinhighereukaryotesincludingplants,mouseandhumancells.m6AplayscrucialrolesinregulatingDNAreplication,DNAdamage,RNAsplicing,transposition,transcription,andcellulardefense.Inhumancells,them6AmodificationisprobablycatalyzedbyamethyltransferasecomplexMETTL3/METTL14andremovedbytheα-ketoglutarate(α-KG)-andFe2+-dependentdioxygenasessuchasFTO,ALKBH5andTET-likeenzymes.ItwasshownthatMETTL3andα-KG/Fe2+-dependentdioxygenasesplayimportantrolesinmanyBIOLOGicalprocesses,rangingfromdevelopmentandmetabolismtofertility.Thedynamicandreversiblechemicalm6AmodificationonDNA/RNAmayalsoserveasanovelepigeneticMarkerofprofoundbiologicalsignificance.Down-regulationofm6Amodificationwasfirstcharacterizedinhumancancercellsandtissues,relativetotheirnormalcontrols.m6AisfoundtobethemostregulatedDNAmodificationincancers.Inadditiontotheregulationincancercells,relativetotheprimarycell/tissueswhichcontainquitelowDNAm6A(<0.001%),ahundreds-foldincreaseofm6Amodificationwasfoundforinvitroculturedhumancells(0.03%-0.22%).
Principle&Procedure
Thiskitisdesignedformeasuringtotalm6Ademethylaseactivity/inhibition.Inanassaywiththiskit,theuniquem6Asubstrateisstablycoatedonthestripwells.Activem6Ademethylasesbindtoanddemethylatem6Acontainedinthesubstrate.Theun-demethylatedm6Ainthesubstratecanberecognizedwithahighaffinitym6Aantibodyandtheimmuno-signalisenhancedwithenhancersolution.Theratiooramountofun-demethylatedm6A,whichisinverselyproportionaltoenzymeactivity,canthenbecolorimetricallyquantifiedthroughanELISA-likereaction.
StartingMaterials
Inputmaterialscanbenuclearextractsorpurifiedenzymes.Theamountofnuclearextractsforeachassaycanbe2µgto20µgwithanoptimalrangeof5µgto10µg.Theamountofpurifiedm6ADNAdemethylasescanbe20ngto1µgwithanoptimalrangeof50ngto500ng,dependingonthepurityandcatalyticactivityoftheenzymes.
WB(10XWashBuffer)
DB(DemethylaseBuffer)
MS(10Xm6ASubstrate)*
BS(BindingSolution)
AS(AssayStandard,2µg/ml)*
CA(CaptureAntibody,1000µg/ml)*
DA(DetectionAntibody,400µg/ml)*
ES(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!
