Description
Non-PEGylateddoxorubicinliposomecomesinathree-vialkitandhasalipidcompositionandliposomesizesimilartocommercialMyocet®.Doxorubicindrugcannotbepre-loadedtotheliposomesinthesamewaythatispre-loadedintothePEGylatedliposomes.ThematrixliposomeinPEGylatedliposomeishydrogenatedsoyPCwhichisasaturatedlipidandcreatesatightpackedliposomesuitableforpre-loADIngandholdingthedruginsidetheliposomes.ThematrixlipidinthisformulationisL-⍺-phosphatidylcholinewhichisanunsaturatedlipid.Itmakesaloosepackedmembraneandtherefore,itcannotholdthedrugforalongtime.Thedoxorubicindrugshouldbeloadedintotheliposomesmomentsbeforeuse.
Thekitcomesinthreevials.Thefirstvialcontainsaveryconcentratedsolutionofliposomes.TheliposomesaremadeincitratebufferatpH4.ThesecondvialiscomposedofsodiumcarbonateatpH11.4.Addingvial2tovial1makesapHgradientaroundthelipidmembrane.DuetothedifferenceinthepHinsideandoutsideoftheliposomes,therewillbeaconstantfluxofprotonfromtheinsidetotheoutsideoftheliposomes.Duetothisflux,doxorubicinmoleculesmoveintheoppositedirectionfromtheoutsidetotheinsideoftheliposomes.Duringtheremoteloadingprocess,duetopHgradient,thedoxorubicindrugisabletofreelypassintotheliposomeswhereitisboundbyhydrogenandnanocrystalsofdoxorubicincitratesalttoformlargerdoxorubicincompounds.Asthenewdoxorubicincomplexesareformed,theycometogethertoformoneofthethreedifferentshapesinsidetheliposomes.Alongsingularbandthatstretchesthemembrane,aclosedcircularbandoranopenedU-shapedband.ThecircularandU-shapedcomplexesarethemostcommonones.DoxorubicinliposomesthatareformedbyremoteloadingusingpHgradientshouldbeusedimmediately.
PEGylatedformulationofdoxorubicinliposomesisalsoavailable.Formoreinformationseehere.
FormulationInformation
Doxosome™Kit-DoxorubicinLiposomes(Non-PEGylated)
| 3-VialKitforDoxosome™(Non-PEGylated) | Specification |
|---|---|
| Vial1 | PreformedliposomescomposedofHSPCandcholesterol(55:45molarratio) |
| Vial2 | SodiumcarbonateformakingapHgradient |
| Vial3 | DoxorubicinHClin0.9%saline |
| Instructions:Add0.4mloftheliposome(vial1)to0.6mlofthesodiumcarbonatesolution(vial2).Thenadd10mgofthedoxorubicinin4mlofthe0.9%salinesolution(vial3)tothemix,heatat55-60°Cfor7min,andshakeitfor10s.Thefinalconcentrationofdoxorubicinintheliposomesis2mg/mlandthetotalvolumeofthereagentis5ml.Over95%ofthedrugisloadedtotheliposomes.Thedrugtolipidw/wratiois0.27:1. | |
| LipidCompositionforVial1 | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 100mg/ml | 166.8mM | 100 |
| L-alpha-Phosphatidylcholine | 71.3 | 92.6 | 55 |
| Cholesterol | 28.7 | 74.2 | 45 |
| BufferandLiposomeSizeforVial1 | Specification |
|---|---|
| Buffer | CitrateBuffer |
| pH | 4(in300mlbuffer) |
| LiposomeSize | 180nm |
| Vial2 | Specification |
|---|---|
| SodiumCarbonate | 166mM |
| pH | 11.4 |
| Vial3 | Specification |
|---|---|
| DoxorubicinHCl | Solutionin0.9%Saline |
LiposomeParticleCalculator
Doxorubicinliposomesareunilamellarandsizedto100nm.Themolarconcentrationofliposomeis166.8mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.
Appearance
Doxosome™isaredtranslucentliquidmadeofnanosizeunilamellarliposomes.Usually,duetothesmallsizeofliposomes,nosettlingwilloccurinthebottomofthevial.Theliposomesarepackagedinanambervial.
EducationalVideo
Ordering/ShippingInformation
- Allliposomebasedformulationsareshippedonblueiceat4°Cininsulatedpackagesusingovernightshippingorinternationalexpressshipping.
- LiposomesshouldNEVERbefrozen.Icecrystalsthatforminthelipidmembranecanrupturethemembrane,changethesizeoftheliposomesandcausetheencapsulateddrugtoleakout.Liposomesinliquidformshouldalwaysbekeptintherefrigerator.
- ClientswhoorderfromoutsideoftheUnitedStatesofAmericaareresponsIBLefortheirgovernmentimporttaxesandcustomspaperwork.EncapsulaNanoSciencesisNOTresponsibleforimportationfeestocountriesoutsideoftheUnitedStatesofAmerica.
- WestronglyencouragetheclientsinJapan,Korea,TaiwanandChinatoorderviaadistributor.Toughcustomsclearanceregulationsinthesecountrieswillcausedelayincustomclearanceoftheseperishableformulationsifordereddirectlythroughus.Distributorscaneasilyclearthepackagesfromcustoms.Toseethelistofthedistributorsclickhere.
- ClientsorderingfromuniversitiesandresearchinstitutesinAustraliashouldkeepinmindthattheliposomeformulationsaremadefromsyntheticmaterialandtheformulationsdonotrequirea“permittoimportquarantinematerial”.LiposomesareNOTBIOLOGicalproducts.
- Ifyouwouldlikeyourinstitute’sFedExorDHLaccounttobechargedforshipping,thenpleaseprovidetheaccountnumberatthetimeofordering.
- EncapsulaNanoScienceshasnocontroloverdelaysduetoinclementweatherorcustomsclearancedelays.YouwillreceiveaFedExorDHLtrackingnumberonceyourorderisconfirmed.ContactFedExorDHLinadvanceandmakesurethatthepaperworkforcustomsisdoneontime. AllsubsequentshippinginquiriesshouldbedirectedtoFederalExpressorDHL.
StorageandShelfLife
Storage
Doxosome™productsshouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.IfthesUSPensionisfrozen,theencapsulateddrugcanbereleasedfromtheliposomesthuslimitingitseffectiveness.Inaddition,thesizeoftheliposomeswillalsochangeuponfreezingandthawing.
ShelfLife
Doxosome™ismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin4monthsofthemanufacturingdate.
Referencesandbackgroundreading
1.HermansonGT.Bioconjugatetechniques.Academicpress;2013Jul25.
2.TorchilinV,WeissigV,editors.Liposomes:apracticalapproach.OxfordUniversityPress;2003Jun5.
3.GrabarekZ,GergelyJ.Zero-lengthcrosslinkingprocedurewiththeuseofactiveesters.Analyticalbiochemistry.1990Feb15;185(1):131-5.
4.YanL,CraytonSH,ThawaniJP,AmirshaghaghiA,TsourkasA,ChengZ.ApH‐ResponsiveDrug‐DeliveryPlatformBasedonGlycolChitosan–CoatedLiposomes.Small.2015Oct1;11(37):4870-4.
5. Silva-LópezEI,EdensLE,BardenAO,KellerDJ,BrozikJA.ConditionsforliposomeadsorptionandbilayerformationonBSApassivatedsolidsupports.Chemistryandphysicsoflipids.2014Oct31;183:91-9.
6.HazraM,SinghSK,andRayS.SurfaceModificationofLiposomalVaccinesbyPeptideConjugation.JournalofPharmaSciTech,2011;1(1):41-47.
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转录的只是插入的片断，怎么会跟模板一样大呢，一般情况下应该比模板小多了。我都是跑电泳，严格来说应该跑变性的；嫌麻烦可以非变性，快速（防止RNA降解）十分钟即可；抛出来的带会有两条，上面大的是模板，下面一条比较亮的是RNA，那么你的转录就 成功了。
PCR第一个步骤如果没记错的话应该叫“变性”， PCR的话是不需要解旋酶的，直接利用DNA的高温变性特性，在高温下，使氢键断开，DNA双链恢复为单链。 （还有解旋酶好像是解除双螺旋的结构，是一种拓扑异构酶，对于氢键的打开没有贡献吧
首先，不否认它比大多数国产的试剂盒做得好，提取的纯度、方便度和量，都还很不错。
其次，它是日本的。so我觉得实验室用用天根的，就非常好了。拒绝日货，从实验室做起。
对于新手来说购买反转录试剂盒是比较理想的，买一个kit看看说明书操作就可以了。推荐两款kit TAKARA和HaiGene，这两款试剂盒都能对RNA样品中的gDNA有效去除，因此对RNA质量的要求不高。TAKARA的RR047A操作方便，反转录温度为37℃，对于大多数试验来讲是满足要求的。HaiGene的D0401操作要多一个步骤，但其反转录温度是55℃（耐高温的反转录酶），提高了反转录温度使得高GC含量、复杂模板、长mRNA的模板都能有效反转录，因此其反转录效率更高，更能够获得样本中基因的真实表达量。如果后续试验是RealTime PCR、ORF克隆、高GC含量、或者你的待研究基因结构复杂程度未知，还是选用耐高温的反转录酶更理想。对于反转录高手来说，直接购买反转录酶、再购买Rnase Inhibitor自己配制反转录体系就可以了。对于样本量大的课题组来讲，相对还是比较经济的。选择反转录酶时仅需要考虑是否需要耐高温的酶，来克服目的基因的复杂结构就可以了。PROMEGA、TAKARA、HaiGene、 TRANSGEN等品牌的反转录酶性价比还都是不错的。Life、NEB的也不错，不过性价比一般，不一一解释了。
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