| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | 266.21 |
| CAS# | 55750-62-4 |
| Solvent | DMSO |
| Storage | F/D |
| Category | ProteinBiochemistry Generalproteins |
| Related | AminetoThiol Thiol-ReactiveProbes BiochemicalAssays |
Generally,a10-to50-foldmolarexcessofSMCCorSMCCPlus™overtheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.
1.Prepareconjugationbufferforamine-containingprotein,Protein-NH2(BufferA):Phosphatebufferedsaline(PBS,pH7.2)orotheramine-freebufferswithpHfrom7.2to8.0areOKtouseforthispurpose.
Note:Avoidbufferscontainingprimaryamines(e.g.,Trisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasPBS.
2.Prepareconjugationbufferforsulfhydryl-containingprotein,Protein-SH(BufferB):Sulfhydryl-freebuffersatpHfrom6.5to7.5areOKtouseforthispurpose.
Note1:Avoidbufferscontainingsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasPBS.
Note2:Theadditionof1-5mMEDTAhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingprotein.
3.Preparedesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionby-products.
4.Prepareamine-containingprotein(Protein-NH2)andsulfhydryl-containingprotein(Protein-SH)solutionsintheirConjugationBufferAandBufferB.
5.PrepareProtein1-NH2-SMCC(orSMCCPlus™)conjugate:AddtheappropriateamountofSMCC(orSMCCPlus™)totheamine-containingproteinsolutioninBufferA.TheconcentrationoftheProtein-NH2determinesSMCC(orSMCCPlus™)molarexcesstouse.ThesuggestedvolumeofSMCC(orSMCCPlus™)molarexcessesareasfollows,butthebestratiohastobedeterminedexperimentallyusingthetargetproteins:
•Proteinsamples<1mg/mLuse40-80-foldmolarexcess.
•Proteinsamplesof1-4mg/mLuse20-foldmolarexcess.
•Proteinsamplesof5-10mg/mLuse5-to10-foldmolarexcess.
6.Incubatetheabovereactionmixtureatroomtemperaturefor30-60minutesorat4°Cfor2-4hours.
Note1:Tostoptheconjugationreactionbeforecompletion,addbuffercontainingreducedcysteineataconcentrationseveraltimesgreaterthanthatofthesulfhydrylsofProtein-SH.
Note2:Conjugationefficiencycanbeestimatedbyelectrophoresisseparationandsubsequentproteinstaining.
7.RemoveexcessSMCC(orSMCCPlus™)byusingadesaltingcolumnequilibratedwithConjugationBufferA.
8.Combineandmixthiol-containing(Protein-SH)anddesaltedamine-containingprotein(Protein-NH2)SMCC(orSMCCPlus™)conjugateinamolarratiocorrespondingtothatdesiredforthefinalconjugateandconsistentwiththerelativenumberofsulfhydrylandactivatedaminesthatexistonthetwoproteins.
Note:Moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.Forproteins,reducedisulfidebondsusing5mMTCEPatroomtemperaturefor30minutes,followedbytwopassesthroughasuitabledesaltingcolumn.Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.Selectivereductionofhinge-regiondisulfidebondsinIgGcanbeaccomplishedwith2-mercaptoethylamine•HCl(2-MEA).SulfhydrylscanbeaddedtomoleculesusingN-succinimidylS-acetylthioacetate(SATA)or2-iminothiolane•HCl(Traut’sReagent),whichmodifyprimaryamines.
| References&Citations |  CitationExplorer |
N-glycantargetedgenedeliverytothedendriticcellSIGNreceptor
Authors:KevinAnderson,ChristianFernandez,KevinGRice
Journal:Bioconjugatechemistry(2010):1479--1485
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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