PNGaseF,PeptideNGlycosidaseF,N-Glycosidase,N-Glycanase
RecombinantPNGaseFisisolatedfromaE.colistraincontainingacloneoftheElizabethkingiamiricolagene.Thereisnodetectabledifferenceinactivityorspecificactivityoftherecombinantenzymefromthenativeenzyme(E-PNG01).
PNGaseFcleavesasparagine-linked(N-linked)oligosaccharidesfromglycoproteins.PNGaseFdeaminatesasparaginetoasparticacid,leavingtheoligosaccharidesintact.Denaturationincreasestherateofcleavageupto100x.MostnativeproteinscanstillbecompletelyN-deglycosylatedbutincubationtimemustbeincreased.PNGaseFwillremainactiveunderincubationconditionsforatleast72hours.PNGaseFwillnotremoveoligosaccharidescontainingAlpha-(1,3)-linkedcorefucosecommonlyfoundonplantglycoproteins;forthispurpose,usepeptideN-glycosidaseA.
PNGaseFSourcerecombinantPNGaseFgenefromElizabethkingiamiricolainE.coli
EC3.5.1.52
Contents
60µlaliquotofrecombinantPNGaseF(0.3U)in20mMTris-HCl,pH7.5
Includedwith20µLand60µLpacksizes
5xPNGaseFReactionBuffer7.5forPNGaseF–250mMsodiumphosphate,pH7.5
PNGaseFDenaturationSolution–2%SDS,1MBeta-mercaptoethanol
PNGaseFTritonX-100–15%solution
SpecificActivity>25U/mg
Activity5U/ml
Molecularweight36,000daltons
pHrangeforPNGaseF6-10,optimum7.5
PNGaseFSuggestedusage
1.Addupto200µgofglycoproteintoanEppendorftube.Adjustto35µlfinalvolumewithde-ionizedwater.
2.Add10µl5xPNGaseFReactionBuffer7.5and2.5µlofPNGaseFDenaturationSolution.Heatat100˚Cfor5minutes.
3.Cool.Add2.5µlofPNGaseFTritonX-100andmix.
4.Add2.0µlofPNGaseFtothereaction.Incubate3hoursat37˚C.
SpecifictityPNGaseFcleavesasparagine-linked(N-linked)oligosaccharidesfromglycoproteins.PNGaseFdeaminatesasparaginetoasparticacid,leavingtheoligosaccharidesintact.Denaturationincreasestherateofcleavageupto100x.MostnativeproteinscanstillbecompletelyN-deglycosylatedbutincubationtimemustbeincreased.PNGaseFwillremainactiveunderincubationconditionsforatleast72hours.PNGaseFwillnotremoveoligosaccharidescontainingAlpha-(1,3)-linkedcorefucosecommonlyfoundonplantglycoproteins;forthispurpose,usepeptideN-glycosidaseA.
SpecificActivityDefinedastheamountofenzymerequiredtocatalyzethereleaseofN-linkedoligosaccharidesfrom1micromoleofRNaseBin1minuteat37˚C,pH7.5.CleavageismonitoredbySDS-PAGE(cleavedRNaseBmigratesfaster).
StorageStoreenzymeat4˚C.
PNGaseFReferences
–Bayer,E.A.,F.DeMeester,T.KulikandM.Wilchek.Preparationofdeglycosylatedeggwhiteavidin.ApplBiochemBiotech53:1-9(1995)
–Elder,J.H.andS.Alexander.endo-b-N-AcetylglucosaminidaseF:endoglycosidasefromFlavobacteriummeningosepticumthatcleavesbothhigh-mannoseandcomplexglycoproteins.ProcNatlAcadSciUSA79:4540-4544(1982)
–Tarentino,A.L.,C.M.GomezandT.H.Plummer,Jr.Deglycosylationofasparagine-linkedglycansbypeptide:N-glycosidaseF.Biochemistry24:4665-4671(1985)
–TarentinoA.L.andT.H.Plummer.Enzymaticdeglycosylationofasparagine-linkedglycans:purification,properties,andspecificityofoligosaccharide-cleavingenzymesfromFlavobacteriummeningosepticum.MethEnzymol230:44-57(1994)
–TrimbleR.B.andA.L.Tarentino.Identificationofdistinctendoglycosidase(endo)activitiesinFlavobacteriummeningosepticum:endoF1,endoF2andendoF3.EndoF1andendoHhydrolyzeonlyhighmannoseandhybridglycans.JBiolChem266:1646-1651(1991)
–Taga,E.M.,A.WaheedandR.L.VanEtten.Structuralandchemicalcharacterizationofahomogeneouspeptide-N-glycosidasefromalmond.Biochemistry23:815-22(1984)
–TarentinoAL,TrimbleRB,PlummerTH.Enzymaticapproachesforstudyingthestructure,synthesis,andprocessingofglycoproteins.MethodsinCellBIOLOGy:32:111–39(1989)
AnthonyL.,TarentinoandThomasH.PlummerJr.Enzymaticdeglycosylationofasparagine-linkedglycans:Purification,properties,andspecificityofoligosaccharide-cleavingenzymesfromFlavobacteriummeningosepticum.MethodsinEnzymology:230:44-57.(1994)
–TarentinoAL,PlummerTH.Oligosaccharideaccessibilitytopeptide:N-glycosidaseaspromotedbyprotein-unfoldingreagents.TheJournalofBiologicalChemistry.257(18):10776–80.(1982)
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1.CA15-3属于多形上皮黏蛋白。CA15-3测定广泛用于乳腺癌的辅助诊断。60%～80％进展期乳腺癌患者的CA15-3浓度大30kU/L。CA15-3有助于判断肿瘤的进展和疗效，测定值大于1000kU/L者预后不良。在乳腺癌初期其灵敏度较低，在Ⅲ期时约为12%，因此CA15-3测定不能用作乳腺癌的筛选手段．当怀疑乳腺癌而CA15-3又正常时，可以结合CEA值进行考虑。应注意到治疗后由于肿瘤细胞死亡，CA15-3起初会升高，然后才逐渐下降。持续不降或上升者则提示病情恶化。
2．其他恶性肿瘤，如肺、结肠、胰腺、肝脏、卵巢癌、子宫颈和子宫内膜的恶性肿瘤也都可见到CA15-3不同程度的阳性率（低于10%）。
3．肝硬化、肝炎、结节病、结核病、自身免疫性疾病（SLE)以及卵巢、乳腺的良性病变中，CA15-3值略高于正常范围，但阳性率一般低于10%，妊娠中后期，有近一半孕妇血清CA15-3升高。
一般情况下同时测定蔗糖、果糖和葡萄糖。
选用哪种检测器呐。
本来经战友的意见已决定用示差了，但突然有个经销商愿意以比较优惠的价格卖蒸发光给我们，所以又开始犹豫了。
最后一次请教各位战友，如题目。谢谢！！
还有如果用气相做糖的鉴别的话，需要样品量大不大？怎么衍生化？
不好意思，我是学药分的，对植化比较外行，请详细些，谢了先！
核酸是天然高分子化合物，，糖类中的多糖（淀粉、纤维素、糖原等）也是天然高分子化合物
同属于生命的六大营养物质（水，无机盐，糖类，脂质，核酸，蛋白质）
暂无品牌问答
