The2-AAlabelingkitcontainstwosetsofthefollowingreagents(15samplesperkit)suppliedinglassampoulessealedunderpurenitrogen: 2-aminobenzoicacid(2-AAdye) Dimethylsulfoxide(DMSO) Aceticacid Sodiumcyanoborohydrideor2-picolineborane
TrADItional2-ABand2-AAlabellingkitsusesodiumcyanoborohydrideasareducingagentduringglycanlabeling.Thisreagentistoxicsoafumecupboardshouldbeusedduringhandling.ToconformwithemerginghealthandsafetyregulationswearenowreplacingthesewithournewVPglycankitsthatusepicolineboranewhichisasignificantlysaferreductant.
NumberofSamplesOne2-AAlabelingkitcontainsreagentstolabelupto30separateanalyticalsamplesperkit.
Dyepurity>99%byHPLC
Molecularweight137
LamBDa-ex320nm
Lambda-em420nm
AmountofSampleFrom25pmolupto25nmolglycanspersample.
SuitableSamplesAnypurifiedglycanswithfreereducingterminicanbelabeled.
StructuralIntegrityNodetectable(<2molepercent)lossofsialicacid,fucose,sulfate,orphosphate.
LabelingEfficiencyTypically>85%(dependentonsample).
LabelingSelectivityEssentiallystoichiometriclabeling.
Protocol1. PurifytheglycansLudgerCleanEB10cartidges(LC-EB10-A6)havebeendesignedforpurificationofglycansfromproteins,salts,anddetergents.
2.TransfersampletoreactionvialTheamountofsampleshouldbeintherange100picomoles–50nanomolesforaglycanpoolobtainedfromatypicalglycoprotein.Withasinglepureglycanaslittleas5picomolescanbelabeledanddetectedinsubsequentHPLCanalysis.SuitablereactionvialsincludesmallpolypropylenemicrocentrifugetubesandtubesforPCRwork.
3. DrythesamplesIdeally,samplesshouldbedriedusingacentrifugalevaporator.IfthisisnotpossIBLethenfreezedrying(lyophilization)canbeusedwithcaution(inparticular,ensurethatthesampledriestoasmall,compactmassattheverybottomofthevial).Donotsubjectsamplestohightemperatures(>28°C)orextremesofpHastheseconditionswillresultinacidcatalysedlossofsialicacids(hightemperatures,lowpH)orepimerizationoftheglycanreducingterminus(athighpH).
4.PrepareaDMSO-aceticacidmixtureAdd150μLglacialaceticacidtothevialofDMSOandmixbyPipetteaction.
5.AddthedyeAdd100μLoftheDMSO-aceticacidmixturetoavialofthe2-AA(2-aminobenzoicacid)dyeandmixuntilthedyeisdissolved.
6.AddthereductantAddthedissolveddyetoavialofsodiumcyanoborohydrideorpicolineboraneandmixbypipetteactionuntilthereductantiscompletelydissolvedtomakethefinallabelingreagent.
7.Add2-AAlabelingreagenttosamplesAdd5μLoflabelingreagenttoeachdriedglycansample,capthemicrotube,mixthoroughly.
8.IncubatePlacethereactionvialsinaheatingblock,sandtray,ordryovensetat65°Candincubatefor3hours.Inmostcases,theincubationtimecanbeshortenedto2hoursorextendedupto4hourswithoutsignificantlychangingtheoutcomeofthelabelingreaction.
9.CentrifugeandcoolAftertheincubationperiodremovethesamples,centrifugethemicrotubesbriefly,thenallowthemtocoolcompletelytoroomtemperature.
10.SampleCleanupPost-labelingsamplecleanupisrecommendedtoremoveexcessdyeandotherlabelingreagents.CleanupcanbeachievedusingLudgerCleanT1cartridges(LC-T1-A6)orScartridges(LC-S-A6)
2-AALabelingReferences
Anumula,K.R.Quantitativedeterminationofmonosaccharidesinglycoproteinsbyhigh-performanceliquidchromatographywithhighlysensitivefluorescencedetection.AnalyticalBiochemistry220:275-283(1994)
Anumula,KR;Dhume,STHighresolutionandhighsensitivitymethodsforoligosaccharidemappingandcharacterizationbynormalphasehighperformanceliquidchromatographyfollowingderivatizationwithhighlyfluorescentanthranilicacid.GlycoBIOLOGy8:685-694(1998)
Anumula,KR;Du,PCharacterizationofcarbohydratesusinghighlyfluorescent2-aminobenzoicacidtagfollowinggelelectrophoresisofglycoproteins.AnalyticalBiochemistry275:236-242(1999)
Bigge,JC;Patel,TP;Bruce,JA;Goulding,PN;Charles,SM;Parekh,RBNonselectiveandefficientfluorescentlabelingofglycansusing2-aminobenzamideandanthranilicacid.AnalyticalBiochemistry230:229-238(1995)
Frears,ER;Merry,AH;Axford,JSScreeningneutralandacidicIgGN-glycansbyhighdensityelectrophoresis.GlycoconjugateJournal16:283-290(1999)
Huang,Z;Prickett,T;Potts,M;Helm,RFTheuseofthe2-aminobenzoicacidtagforoligosaccharidegelelectrophoresis.CarbohydrateResearch328:77-83(2000)
Sato,K;Sato,K;Okubo,A;Yamazaki,SDeterminationofmonosaccharidesderivatizedwith2-aminobenzoicacidbycapillaryelectrophoresis.strong>AnalyticalBiochemistry251:119-121(1997)
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一般来讲,进行real-time qPCR MasterMix都是2×的浓缩液,只需要加入模板和引物就可以。由于real-time qPCR灵敏度高,所以每个样品至少要做3个平行孔,以防在后面的数据分析中,由于Ct相差较多或者SD太大,无法进行统计分析。通常来讲,反应体系的引 物终浓度为100-400mM;模板如果是总RNA一般是10ng-500,如果cDNA,通常情况下是1ul或者1ul的10倍稀释液,要根据目的基因 的表达丰度进行调整。当然这些都是经验值,在操作过程中,还需要根据所用MasterMix,模板和引物的不同进行优化,达到一个最佳反应体系。在反应体 系配置过程中,有下面几点需要注意:
1. MasterMix不要反复冻融,如果经常使用,最好溶解后放在4度。
2. 更多的配制Mix进行,减少加样误差。最好能在冰上操作。
3. 每管或每孔都要换新枪头!不要连续用同一个枪头加样!
4. 所有成分加完后,离心去除气泡。
5. 每个样品至少3个平行孔。
设计方法:通用茎环后端加miRNA后面6个碱基的反向互补序列为反转录引物,正向引物为miRNA去除后面6个碱基的序列,反向引物在茎环上。
反转录茎环通用序列:
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAC
miRNA序列:
>mmu-miR-99b-5p
MIMAT0000132 Mus musculus miR-99b-5p
CACCCGUAGAACCGACCUUGCG
mmu-miR-99b-5p反转录颈环引物为:
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCGCAAG
定量PCR反向引物为:TGTCGTGGAGTCGGC
正向引物为:CACCCGTAGAACCGAC
在分子生物学中,探针是根据碱基互补的原理,用来与特定的DNA片段做杂交以对特定的DNA片段进行检测,如Southern杂交
引物是用来扩增DNA序列的,因为核苷酸必须要连接在3-OH上才能够合成延伸,引物就是用来提供3-OH的。
只要是符合要求的DNA片段都可以用来做探针和引物,引物是用来扩增DNA序列,探针用来与特定的DNA片段做分子杂交的
上面较高的是样本曲线,下面较低的是未加样本(茎环引物和引物、探针都加入)
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