| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 346/449 |
| MW | ~400 |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes iFluor™DyesandKits |
| Related | Carbonyl-ReactiveProbes OtherProteinModifications |
| Spectrum | AdvancedSpectrumViewer |
SampleNeuronCellLabelingProtocol
ThissamplelabelingprotocolwasdevelopedforlabelingneuroncellswithiFluor™488hydrazide.Youmightneedfurtheroptimizationforyourparticularcells.
1. Preparedyestocksolution:
a. Dissolve1mgofiFluor™488hydrazidein100µlof200mMKCltomake10mMsolution(10mg/ml).
b. Sterilizetheabovesolutionbyfiltration.
2. LabelCells:
a. Labelneuroncells(e.g.,accessoryplantaretractormotoneuronsinManducasextalarvae)withtheiFluor™488hydrazidestocksolution(fromStep1)bypassinghyperpolarizingcurrentthroughtheelectrode.
3. Fixcells:
a. Fixthecellswith4%paraformaldehydein100mMphosphatebuffer(pH7)for30min.
b. Note:Followingfixation,thecellsmaybedehydratedinethanolandclearedinxylene.
SampleGlycoproteinLabelingProtocol
ThislabelingprotocolwasdevelopedfortheconjugationofGoatanti-mouseIgGwithiFluor™647hydrazide.Youmightneedfurtheroptimizationforyourparticularproteins.
1. Prepareproteinaldehydesolution(SolutionA):
a. Dissolve5mgofgoatanti-mouseIgGantibodyin0.5mLof0.1Msodiumacetate,0.15MNaCl,pH5.5
i. Note:Makesureproteinconcentration>10mg/mlforbestlabelingefficiency.
b. Treattheproteinsolutionwith0.2mlof100mMsodiumperiodate(21mg/ml)byshakingfor30-60min.
c. Stopthereactionsbyadding30µLethyleneglycol.
2. Preparedyestocksolution(SolutionB):
a. Dissolve1mgiFluor™dyehydrazidein0.1mlDMSOtomake10mg/mldyestocksolution.Mixwellbypipettingorvortex.
3. Runconjugationreaction:
a. Addthe10µLofdyestocksolution(SolutionB)into50µLoftheproteinsolution(SolutionA)witheffectiveshaking.
b. Continuetorotateorshakethereactionmixtureatroomtemperaturefor30-60minutesinthedark.
4. PurifytheconjugationThefollowingprotocolisanexampleofdye-proteinconjugatepurificationbyusingaSephadexG-25column.
a. PrepareSephadexG-25columnaccordingtothemanufactureinstruction.
b. Loadthereactionmixture(directlyfromStep3)tothetopoftheSephadexG-25column.
c. AddPBS(pH7.2-7.4)assoonasthesamplerunsjustbelowthetopresinsurface.
d. AddmorePBS(pH7.2-7.4)tothedesiredsampletocompletethecolumnpurification.Combinethefractionsthatcontainthedesireddye-proteinconjugate.
Note1:Forimmediateuse,thedye-proteinconjugatemightbealiquotedformultipleuseswhileforlongertermstorage,dyeproteinconjugatesolutionneedbeconcentratedorfreezedried.
Note2:Forsmallscalelabeling,aready-to-usespincommercialcolumnisrecommendedforconvenience.
| References&Citations |  CitationExplorer |
DeepSequencingAnalysisoftheEha-RegulatedTranscriptomeofEdwardsiellatardaFollowingAcidification
Authors:DGao,NLiu,YLi,YZhang,GLiu
Journal:Metabolomics(LosAngel)(2017):2153--0769
Suramininhibitscullin-RINGE3ubiquitinligases
Authors:KennethWu,RobertAChong,QingYu,JinBai,DonaldESpratt,KevinChing,ChanLee,HaibinMiao,IngerTappin,JerardHurwitz
Journal:ProceedingsoftheNationalAcademyofSciences(2016):E2011--E2018
GlycosaminoglycanmimicrybyCOAMreducesmelanomagrowththroughchemokineinductionandfunction
Authors:HelenePiccard,NeleBerghmans,EvaKorpos,ChrisDillen,IlseVanAelst,SandraLi,ErikMartens,SandraLiekens,SamNoppen,JoVanDamme
Journal:InternationalJournalofCancer(2012):E425--E436
