Ficoll PM 400 聚蔗糖 PM 400

ProductDescription

Ficoll400isahighlybranchedpolymerformedbythecopolymerizationofsucroseandepichlorohydrin.Ficoll400iscompletelynon-ionic.Becauseoftheabundanceofhydroxylgroups,Ficoll400isveryhydrophilicandextremelywater-soluble.ThemostcommonapplicationforFicoll400isasadensitygradientmediumfortheseparationandisolationofeukaryoticcells,organellesandbacterialcells.Densityrangesupto1.2g/mlcanbeattained.Ithasalsobeenutilizedinavarietyofotherapplications.

PhysicalProperties

Appearance:Whitetowhitewithfaintyellowcast

PowderLossondrying:Notmorethan5%1MolecularWeight:400,000+/-100,000asdeterminedbyintrinsicviscosity1SpecificRotation:+56.5°at20°C(C=1%inwater)1Intrinsicviscosity:approximately0.17dl/g1DialyzablematerialincludingNaCl:lessthan1%1StokesRadius:approximately10nm1

Unlikesucrose,solutionsofFicollhaverelativelylowosmolality.Despitethis,Ficollsdensityinaqueoussolutionsiscomparabletothatofsucrose.Becauseofthehighmolecularweightandlowcontentofdialyzablematerial,Ficollhasamuchlowerpermeabilitytowardscellmembranesthansucrose.Therefore,cellscanbeexpectedtocollectatalowerdensityinFicollgradientsthaninsucrosegradients.

Becauseofitslowmembranepermeabilityandlowosmoticpressure,separationsinFicollnormallyresultinbetterpreservationofcellfunctionandmorphology.

Storage/Stability

StoredproperlyasapowderatroomtemperatureFicoll400canbeexpectedtohaveashelf-lifeof5years.

Solubility/Solutionstability

Concentrationsof50%(w/v)canbeattainedinwater.Ficollshouldbeaddedslowlywithconstantstirring.SigmateststhesolubilityofFicoll400at1gin10mlofdeionizedwateryieldingacleartoslightlyhazy,colorlesstofaintyellowsolution.Ficollisstableinalkalineandneutralsolutions.AtpHvaluesbelow,itisrapidlyhydrolyzed,particularlyatelevatedtemperatures.FicollcanbesterilizedbyautoclavingataneutralpH,at110°Cfor30minutes.Strongoxidizingandreducingagentsaretobeavoided.

Procedure

Centrifugation

Ficoll400canbeusedforgradientcentrifugationinalltypesofcentrifugerotorsandforseparationatunitgravity.Forcentrifugation,bothdiscontinuousandcontinuousgradientsarepossible.Discontinuousgradientsoffertwomainadvantages:First,theabruptchangesinFicoll400densitymeanthatisolatedcellsarefoundinsharpbandsattheinterfacebetweenlayersofdifferentdensities.Thisallowsforeasyremovalofthesamplewithapipette.Second,cellswithgreatdifferencesindensitycanbeeasilyisolatedwithasfewastwodensitylayers.Thisisachievedbychoosingdensitieswhichwillpreventoneormorecelltypesfromenteringthelowerphase,bandingthesecelltypesattheinterface.Toestimatethedensitiesrequiredforaparticularapplication,consultthetablebelow.

SourceBuoyantDensityConditionsMembranes1.05100,000xgfor16hrsChromatophores1.07195,000xgfor36hrsHepatocytes1.10-1.156,000xgfor2hrsFibroblasts1.058,000xgfor1hrEhrlichascitescells1.071,400xgfor45min

ThegraphofdensitiesofFicollasafunctionofconcentrationfollows.

Preparingadiscontinuousgradient

1.PrepareFicoll400inbufferorisotonicsucrosesolution(0.25M)atconcentrationswhichshouldseparatethematerialofinterest.Mostcellsandorganelleshaveabuoyantdensitybetween1.0and1.2g/mlinFicoll400.Oftenatwo-layergradientissufficient.Solutionsmadeatthisstepmaybestoredintherefrigerator,butshouldbeusedatroomtemperature.

2.Instandardcentrifugetubes,makelayers(approximately1cmdeep)withthemostdenselayeronbottom.

3.Takingcarenottomixthelayersofthegradient,carefullylayerthesampleontop.StirthesampleandtheuppermostFicoll400layergentlywithaglassrodtoeliminatetheinterfacebeforecentrifugation.

DuringcentrifugationthevariousparticleswillcollecteitherinorbetweentheFicolllayers,dependingontheirdensity.Uponcompletionofcentrifugation,pipetteoffthevariousphasesandremovetheFicollfromthefractionsofinterest.Ficollmayberemovedfromisolatedcellsandorganellesbyrepeatedcyclesofdilutionwithbufferfollowedbycentrifugation.ResidualamountsofFicoll400inthesamplecanbeestimatedwiththeanthronereaction

Insomeinstances,acontinuousorlineardensitygradientmaybedesired.Thiscanbeeasilypreparedusingagradientmixer.Forsimpleseparations,ahomologousFicoll400solutionwithoutagradientcanbeused.Fractionationisaccomplishedbystepwiseincreasesincentrifugationspeed.Ficollhasalsobeenemployedinzonalcentrifugationstudies.

Unitgravitysedimentationthroughadensitygradientiswidelyusedtoseparatecellswhicharesensitivetocentrifugation.Cellswithsimilardensitiesbutdifferentsizescanalsobeefficientlyseparatedatunitgravity.

Ficoll-HypaqueDensityMedia(Histopaque)

ApplicationscallingforFicoll-HypaqueemployamixtureofFicollandsodiumdiatrizoate.SigmaofferspreformedmixturesofthreedifferentdensitiesundertheproductnamesofHistopaque-1077,1083and1119.

NucleicAcidHybridization

Ficoll400isaconstituentofDenhardt’ssolutionusedinNorthernandSouthernblotanalysis.Ficollreducesnon-specificbindingofmaterialtonitrocellulosemembranesduringnucleicacidhybridization.SigmaoffersaDenhardt’ssolution(ProductNumberD2532),50Xconcentrate,testedforuseinnucleicacidhybridization.Typicalhybridizationsolutionsrequirea5XconcentrationofDenhardtssolution.

ImmunologicalApplications

Ficoll400hasbeenemployedasahaptencarrier,andhasbeenconjugatedtodinitrophenol,trinitrophenol,andfluoresceinisothiocyanateforthepurposeofenhancingprimaryimmuneresponseinmice.Conjugateswitharangeofsubstitutionlevelsandminimaltoxicityareeasilyprepared.

ChemicallyDefinedCellCultureMedia

Ficollisusedwithandwithoutserum-derivedgrowthfactorstosupportthegrowthofbothprimaryculturesandestablishedcelllines.

ConcentrationDialysis

Ficoll400isusefulforconcentratingsolutionsbydialysis,sinceitshighmolecularweightpreventsitfromcrossingthedialysismembrane.OsmoticpressuredrawswateracrossthemembraneintothesolutionofFicoll400,effectivelyconcentratingsensitivematerials.

Electrophoresis

Continuousflowelectrophoresisusuallyrequiresastabilizerintheelectrolyte.Ficoll400isoftenusedforthisapplication.

PhasePartitioning

Phasepartitioningseparatescellsonthebasisofsurfaceproperties.Ficoll400iscombinedwithpolyethyleneglycolintwo-phasesystems,andwithdextranandpolyethyleneglycolinthreephasesystems.

PhysiologicalPerfusionandCellStabilizationSolutions

Ficollhasbeenaddedtophysiologicalsalineperfusateduringmonitoringofproteinexcretioninvessels.Vitrifiedmouseembryoshavebeendilutedwithsolutionscontaining30%Ficollplus0.5Msucrose.IsolatedratkidneyswereperfusedwithTyrode’ssolutioncontaining4.7%Ficoll400.18

*FicollisaregisteredtrademarkofPharmacia.InformationonphysicalpropertiesandapplicationswasobtainedfromPharmacia.

货号	名称	英文名称	规格	单价
Sigma-F4375	聚蔗糖PM400	FicollPM400	10G	445