| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 565/600 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related |
| Spectrum | AdvancedSpectrumViewer |
- Prepareantibodysolution:
Forlabeling100μgantibody(assumingthetargetantibodyconcentrationis1mg/mL),mix5μL(5%ofthetotalreactionvolume)ofReactionBuffer(ComponentC)with100μLofthetargetantibodysolution.
Note1.Ifyouhaveadifferentantibodyconcentration,adjusttheantibodyvolumeaccordinglytomake~100µgantibodyavailableforyourlabelingreaction.
Note2:Theantibodyshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Iftheantibodyisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note3:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.
Note4:TheAntibody–Buccutite™MTAreactionefficiencyissignificantlyreducediftheantibodyconcentrationislessthan1mg/mL.Foroptimallabelingefficiencythefinalantibodyconcentrationrangeof1-10mg/mLisrecommended. - RunAntibody-Buccutite™MTAreaction:
- AddtheantibodysolutiondirectlyintothevialofBuccutite™MTA(ComponentB),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
- KeeptheAntibody-Buccutite™MTAreactionmixtureatroomtemperaturefor30-60minutes.
Note:TheAntibody-Buccutite™MTAreactionmixturecanberotatedorshakenforlongertimeifdesired.
- PreparespincolumnforAntibody-Buccutite™MTApurification:
- Inverttheprovidedspincolumn(ComponentD)severaltimestore-sUSPendthesettledgelandremoveanybubbles.
- Snapoffthetipandplacethecolumninawashingtube(2mL,notprovided).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).
- Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
- Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor2minutestoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
- Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
- PurifytheAb-Buccutite™MTAsolution:
- Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,notprovided).Carefullyloadthesample(~105μL,fromStep2.2)directlytothecenterofthecolumn.
- AfterloADIngthesample,add5μLof1XPBS(pH7.2-7.4)tomakethetotalvolumeof110μL.Centrifugethecolumnfor5-6minutesat1,000xg,andcollectthesolutionthatcontainsthedesiredprotein-Buccutite™MTAsolution.
- MakeAb-PEorPETandemconjugation:
- MixwholevialofBuccutite™FOL-ActivatedPEorPETandem(ComponentA)withthepurifiedAb-Buccutite™MTAsolution(fromStep4.2),androtatethemixturefor1houratroomtemperature.
- TheAb-PEorPETandemconjugateisnowreadytouse.
Note1:Forimmediateuse,theAb-PEorPETandemconjugateneedbedilutedwiththebufferofyourchoice.
Note2:Theconcentrationoftheconjugateisabout0.5~0.6mgAb/mLifstartwith100uL1mg/mlantibodysolution.
| References&Citations |  PrinterFriendlyVersion |
1. ZhaoKH,SuP,LiJ,TuJM,ZhouM,BubenzerC,ScheerH.(2006)Chromophoreattachmenttophycobiliproteinbeta-subunits:phycocyanobilin:cysteine-beta84phycobiliproteinlyaseactivityofCpeS-likeproteinfromAnabaenaSp.PCC7120.JBiolChem,281,8573.
2. PetrasekZ,SchmittFJ,TheissC,HuyerJ,ChenM,LarkumA,EichlerHJ,KemnitzK,EckertHJ.(2005)ExcitationenergytransferfromphycobiliproteintochlorophylldinintactcellsofAcaryochlorismarinastudiedbytime-andwavelength-resolvedfluorescencespectroscopy.PhotochemPhotobiolSci,4,1016.
3. LoosD,CotletM,DeSchryverF,HabuchiS,HofkensJ.(2004)Single-moleculespectroscopyselectivelyprobesdonorandacceptorchromophoresinthephycobiliproteinallophycocyanin.BiophysJ,87,2598.
4. PrasannaR,PrasannaBM,MohammadiSA,SinghPK.(2003)EvaluationofTolypothrixgermplasmforphycobiliproteincontent.FoliaMicrobiol(Praha),48,59.
5. PrasannaR,DharDW,DominicTK,TiwariON,SinghPK.(2003)IsolationandcharacterisationofphycobiliproteinrichmutantofcyanobacteriumSynechocystissp.ActaBiolHung,54,113.
6. WuP.(2000)[Phycobiliproteinandfluorescenceimmunologicalassay].ShengLiKeXueJinZhan,31,82.
7. NoubirS,LuqueI,OchoadeAldaJA,PerewoskaI,TandeaudeMarsacN,CobleyJG,HoumardJ.(2002)Co-ordinatedexpressionofphycobiliproteinoperonsinthechromaticallyadaptingcyanobacteriumCalothrixPCC7601:aroleforRcaDandRcaG.MolMicrobiol,43,749.
8. TingCS,RocapG,KingJ,ChisholmSW.(2001)PhycobiliproteingenesofthemarinephotosyntheticprokaryoteProchlorococcus:evidenceforrapidevolutionofgeneticheterogeneity.MicroBIOLOGy,147,3171.
9. TriantafilouK,TriantafilouM,WilsonKM.(2000)Phycobiliprotein-Fabconjugatesasprobesforsingleparticlefluorescenceimaging.Cytometry,41,226.
10. ZhaoKH,DengMG,ZhengM,ZhouM,ParbelA,StorfM,MeyerM,StrohmannB,ScheerH.(2000)Novelactivityofaphycobiliproteinlyase:boththeattachmentofphycocyanobilinandtheisomerizationtophycoviolobilinarecatalyzedbytheproteinsPecEandPecFencodedbythephycoerythrocyaninoperon.FEBSLett,469,9.
AAT Bioquest AAT Bioquest是一家位于美国的生物公司，前身为ABD Bioquest，总部位于加利福尼亚州。专门从事光学检测技术十多年，一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术，综合了化学、生物学和信息学等各个领域的研究，引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络，为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。
美国AATBioquestInc.(前身是ABDBioquest，Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域，包括吸收（颜色），荧光和发光技术。AATBioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学，免疫学，细胞生物学和分子生物学等领域。AATBioquest会不断介绍新产品，快速的丰富各个领域的产品。
1）我们提供反应荧光探针和发光探针，生物素和端粒酶能够应用于标记药物小分子和生物聚合物，如蛋白、核酸以及其他碳水化合物；2）我们研究并生产荧光和发光探针用于检测蛋白，核酸和活细胞。3）我们不断的推出新型的荧光和发光探针用于检测多种酶，特别是检测水解酶和氧化还原酶类；4）我们致力于开发用于信号转导研究的试剂；5）我们提供生理和神经探针，特别是钙离子指示剂和膜电位探针。
作为AATBioquestInc.的中国区域代理，艾美捷科技为中国客户提供光谱学检测领域，包括吸收（颜色），荧光和发光技术等全系列解决方案。我们也将一如既往更加努力为国内用户提供快捷、方便的高质量产品，同时更为您售前售后全面技术支持。
AATBioquest,Inc.(formerlyABDBioquest,Inc.)develops,manufacturesandmarketsbioanalyticalresearchreagentsandkitstoscientistsengagedinlifesciencesresearch,diagnosticR&Danddrugdiscovery.Wespecializeintheareaofphotometricdetectionsincludingabsorption(color),fluorescenceandluminescencetechnologies.TheCompany"sproductsenablescientistsandbiomedicalresearcherstobetterunderstandbiochemistry,immunology,cellBIOLOGyandmolecularbiology.AATBioquestconstantlyintroducesnewproducts,andoffersarapidlyexpandinglistofproductsthataregroupedintoseveralproductlines.
1)Ourreactivefluorescentandluminescentprobes,biotinsandtagenzymesareusedforlabelingsmalldrugmoleculesandbiopolymers,e.g,proteins,nucleicacidsandcarbohydrates;2)Wedevelopfluorescentandluminescentprobesfordetectingproteins,nucleicacidsandlivecells;3)Weconstantlyintroducenovelfluorescentandluminescentprobesfordetectingvariousenzymes,inparticular,hydrolyticandredoxenzymes;4)Wefocusondevelopingreagentsforsignaltransductionresearch;and5)Wealsoofferphysiologicalandneurologicalprobes,e.g.,calciumindicatorsandmembranepotentialprobes.
Besidesthestandardcatalogproductswealsooffercustomservicetomeetyourspecialresearchneeds.Ourcurrentservicesincludecustomsynthesisofcolorimetric,fluorescentandluminescentprobes,customdevelopmentofbiochemical,cell-basedanddiagnosticassaysandcustomscreeningofyourcompoundlibrariesagainstyourdefinedtargetsusingourvalidatedHTSassays.
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不知道发在这里合适不，实在是求助无门啊！版主手下留情。
一、探针DNA标记方法
步骤 ：
1．10ng~3ugDNA每管，双蒸水定量至总体积15ul
2．沸水水浴10分钟后迅速冰浴
3．加入 5号试剂 2ul ， 6号试剂 2ul ，7号试剂 1ul
4．37度1小时~20小时
5．中止反应 加2ul 0.2M EDTA (pH 8.0) 和/或 65度 水浴 10分钟
二、标记效率检测
（一） 试剂配置
Solution Composition Preparation
Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20
Maleic acid buffer 0.1 M Maleic acid, 0.15 M NaCl; adjustwith NaOH (solid) to pH 7.5 (20° C)
Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)
TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Blocking stock solution Dissolve Blocking reagent (bottle 10) 10% (w/v) in Maleic
(10 × conc.) acid buffer underconstantly stirring on a heating block(65°C) or heat in a microwave oven,autoclave. The solution remains opaque
Blocking solution Prepare a 1 × working solution by dilutingthe 10 × Blocking solution 1:10in Maleic acid buffer.
Antibody solution Centrifuge Anti-Digoxigenin-AP(vial 8) for 5 min at 10 000 rpm in theoriginal vial prior to each use, and pipet the necessary amount carefully from thesurface. Dilute Anti-
Digoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution.
Colorsubstrate Add 40 _l of NBT/BCIP (vial 9) to 2 ml of Detection buffer.
solution Note: Store protected from light!
（二）对照的标记DNA（4号试剂）系列稀释
（三）步骤
1、取以上制备的管2~9各1ul，以及自己标记的探针1ul，点到一小片尼龙膜
2、通过紫外线或者120度半小时使核酸交连到膜上
3、膜置塑料盒中加Maleic acid buffer 20ml ，15~25度轻摇孵育2分钟
4、10ml Blocking solution 孵育30分钟
5、10ml Antibody solution 孵育30分钟
6、10ml Washing buffer 洗2次，每次15分钟
7、10ml Detection buffer中平衡2~5分钟
8、膜在2ml新鲜配制的 Colorsubstrate solution 中避光孵育。显色期间避免摇动
9、中止反应 用TE-buffer或者双蒸水洗5分钟
蛋白酶K预处理
三、样品检测与杂交
（一） 步骤
1、 稀释供试品及阳性对照DNA，点膜
2、 通过紫外线或者120度半小时使核酸交连到膜上
3、 将标记好的探针稀释到约25ng/ml, 煮沸5分钟后迅速冰浴
4、膜放入预热好的预杂交液（3.5ml/100cm2）中，充分混匀，避免起泡
5、倒掉预杂交液，加入杂交液及已变性的探针。杂交温度下至少孵育6小时
四、洗膜
1、2 × SSC, 0.1% SDS ， 15-25° C，洗膜2次，每次5分钟。
2、0.5× SSC, 0.1% SDS ，预热至65~68° C，洗膜2次，每次5分钟
五、结果检测
（一） 试剂配制
Solution Composition Preparation
Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20
Maleic acid buffer 0.1 M Maleic acid, 0.15 M NaCl; adjustwith NaOH (solid) to pH 7.5 (20° C)
Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)
TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Blocking stock solution Dissolve Blocking reagent (bottle 10) 10% (w/v) in Maleic
(10 × conc.) acid buffer underconstantly stirring on a heating block(65°C) or heat in a microwave oven,autoclave. The solution remains opaque
Blocking solution Prepare a 1 × working solution by dilutingthe 10 × Blocking solution1:10in Maleic acid buffer.
Antibody solution Centrifuge Anti-Digoxigenin-AP(vial 8) for 5 min at 10 000 rpm in theoriginal vial prior to each use, and pipet the necessary amount carefully from thesurface. Dilute Anti-
Digoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution.
Colorsubstrate Add 40 _l of NBT/BCIP (vial 9) to 2 ml of Detection buffer.
solution Note: Store protected from light!
（二） 步骤
1、Washing buffer洗膜1~5分钟
2、100ml Blocking solution 孵育30分钟
3、20ml Antibody solution 孵育30分钟
4、100ml Washing buffer 洗2次，每次15分钟
5、20ml Detection buffer中平衡2~5分钟
6、膜在10ml新鲜配制的 Colorsubstrate solution 中避光孵育。显色期间摇动
7、中止反应 用TE-buffer或者双蒸水50ml洗5分钟展开
荧光标记物常用的有几十种，比如FITC， PE等等，各个生产厂家还有自己的专利产品
2、 血浆：EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。
3、 细胞上清液：1000×g离心10分钟去除颗粒和聚合物。
4、 组织匀浆：将组织加入适量生理盐水捣碎。1000×g离心10分钟，取上清液。
5、 保存：如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒，检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。
此IBL试剂盒能用于小鼠血清,EDTA血浆,细胞上清中白介素-6的定量检测 试剂盒成分 1 预包被板: 抗小鼠白介素-6兔子IgG,亲合纯化 96T 2 酶标记抗体: (30倍浓缩)HRP标记抗小鼠白介素-6兔子IgG,亲合纯化 0.4mL x 1 3 标准品: 重组小鼠白介素-6 0.5mL x 2 4 EIA缓冲液: 含1% BSA, 0.05%吐温20 BPS 30mL x 1 5 标记抗体稀释液: 含1% BSA, 0.05%吐温20 BPS 12mL x 1 6 显色剂: TMB底物液 15mL x 1 7 终止液: 1N硫酸 12mL x 1 8 浓缩洗涤液: (40倍浓缩) 含1% BSA, 0.05%吐温20 BPS 50mL x 1 操作说明 1实验所需器材(但试剂盒没有提供) 酶标仪(450nm) 微移液管及其吸嘴 量筒及烧杯 去离子水 冰箱(4°C) 坐标纸(log/log) 吸水纸 试管(用于标准品稀释) 温育箱(37°C ± 1°C) 洗瓶 (用于洗板) 一次性试剂管(用于浓缩酶标记抗体和显色剂)向左转|向右转
而放射性核素标记，是对体外诊断试剂的某些元素进行放射性特征性标记，便于检测而已，这类的放射性强度不大，危害不高
