| InputType: | DNA |
| ResearchArea: | DNAMethylation |
| TargetApplication: | AmountQuantitation |
| VesselFormat: | 96-WellPlate |
| 100%Guarantee: | 6months |
The MethylFlash™ GlobalDNA Hydroxymethylation (5-hmC)ELISAEasyKit (Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantifyglobalDNAhydroxymethylationstatusbyspecificallymeasuringlevelsof5-hydroxymethylcytosine(5-hmC)inasimplified,“one-step”ELISA-likeformat.AsafourthgenerationtechnologyofEpigentek"spopularglobalDNAmethylationtechnique,itisafurtherrefinementofthepredecessorMethylFlashkitbyimprovinguponspeed,simplicity,sensitivity,andreproducibility.
Thiskitisalsospecificallyoptimizedforpairedusewiththe MethylFlashGlobalDNAMethylation(5-mC)ELISAEasyKit(Colorimetric) forsimultaneouslyquantifyingbothmethylatedDNAandhydroxymethylatedDNA.
Thiskithasthefollowingadvantagesandfeatures:
- Fast -Reducedstepssothattheentireprocedureonlyneeds2hours*
- Robust -Improvedkitcompositionallowstheassaytohaveagreater“signalwindow”withreducedvariationbetweenreplicates
- Convenient -Inherentlylowbackgroundnoise,therebyeliminatingtheneedforDNAdenaturationandplateblockingsteps
- Sensitive -Detectionlimitcanbeaslowas0.01%hydroxymethylatedDNAfrom100ngofinputDNA
- Specific -Highspecificityto5-hmC,withnocross-reactivitytounmethylatedcytosineormethylatedcytosinewithintheindicatedconcentrationrangeofthesampleDNA
- Universal –PositiveandnegativecontrolsandallowdetectionofDNAhydroxymethylationinanyspeciesfromeithersingle-strandedordouble-strandedinputDNA
- Accurate -Optimizedpositivecontrolsthatcanbefractionalizedinpercentagescale,allowingtheassaytobemoreaccurateandhighlycomparablewithHPLC-MSanalysis
- FlexIBLe -Strip-wellmicroplateformatmakestheassayavailableformanualorhighthroughputanalysis
*Basedonasinglesampleassayinduplicate
Background
5-hydroxymethylcytosine(5-hmC),asasixthDNAbasewithfunctionsintranscriptionregulation,hasbeendetectedtobeabundantinhumanandmousebrainsandembryonicstem(ES)cells.Inmammals,itcanbegeneratedbytheoxidationof5-mC,areactionmediatedbytheten-eleventranslocation(TET)familyof5-mC-hydroxylases. 5-hmChasbeendemonstratedtobetissuespecific,rangingfromundetectablelevelsinculturedcelllinesto0.6%inhumanbraintissues,andcanbeashighas8%oftotalDNAinsomeotherspecies.TheBIOLOGicalsignificanceof5-hmCasanimportantepigeneticmodificationinphenotypeandgeneexpressionhasbeenrecentlyrecognized.Forexample,globaldecreasein5-hmCcontent(DNAhypomethylation)hasbeenexhibitedinnearlyallcancersandhasbeenproposedasamolecularMarkerandtherapeutictargetincanceraswell.
Principle&Procedure
ThiskitcontainsallreagentsnecessaryforthequantificationofglobalDNAhydroxymethylation.Inthisassay,DNAisboundtostrip-wellsthatarespecificallytreatedtohaveahighDNAaffinity.ThehydroxymethylatedfractionofDNAisdetectedusinga5-hmCmAb-baseddetectioncomplexinaone-stepmannerandthenquantifiedcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometer.ThepercentageofhydroxymethylatedDNAisproportionaltotheODintensitymeasured.
StartingMaterials
InputDNAshouldberelativelypurewith260/280ratio>1.6andcanbedilutedwithwaterorTEbuffer.TheDNAamountcanrangefrom20ngto200ngperreaction.However,werecommendusing100ngofDNA,whichistheoptimizedinputamountforthebestresults.
WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControlwith0%5-hmC,50µg/ml)*
PC(PositiveControlcontains1%5-hmC,50µg/ml)*
hmAb(5-hmCAntibody,1000X)*
SI(SignalIndicator,1000X)*
ES(EnhancerSolution,1000X)*
DS(DeveloperSolution)
SS(StopSolution)
8-WellAssayStrips(WithFrame)
UserGuide
*Spinthesolutiondowntothebottompriortouse.
Note:TheNCisunhydroxymethylatedDNAcontaining0%of5-hydroxymethylcytosine.ThePC ishydroxymethylatedDNAcontaining1%of5-hydroxymethylcytosine.
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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!
