Description: -VijaiKrishnanPh.D,LouisianaStateUniversity -HARISHN.RAMANATHANPh.D.,NIDDK,NIH -RadmilaHrdlickova,Ph.D.,UTAustin -DorisBenbrook,Ph.D.,OUHSC Yes,IcertainlydidusethatsampleofPepMuteyougenerouslysentus
anditworkedsowellonHepG2cellsthatIhavesinceorderedafullsizeandamusingitexclusivelyforsiRNA! IcomparedtoRNAiMAXandRochesX-tremeGENEproducts,buttheseproductswerenotaseffectiveasPepMute. Thanksagainforthesample. Itwasfantastic! -MatthewJackson, Ph.D.,USDA
PepMutesiRNATransfectionReagent,formulatedfromsimulationofviruscellpenetratingpeptides(CPPs),isatotalnovelsiRNAdeliverytoolwhichprovidesmorethan95%silencingefficiencyat1nMsiRNAinvarietyofmammaliancells.Withourproprietarypeptidesimulationtechnology(PST),hundredsofviralCPPsweresimulated,synthesizedandscreenedforgenedeliveryefficacyinvarietyofmammaliancells(Figure1).PepMutereagentwasthenidentifiedandvalidatedasanexceptionallyefficientvectorforcondensingandtransfectingshort(under100bp)singleordoublestrandednucleicacidssuchassiRNA,miRNAmimicsandDNAoligoestowidespectrumofmammaliancells.
Figure1.AcartoonshowingPepMutesiRNATransfectionReagentwasdevelopedbyPST
Size:
-PepMuteReagent,1.0mL,sufficientfor~1,333reactionsbasedontransfecting0.5~5.0pmolsiRNAormiRNAmimicsin24-wellplate
-PepMuteTransfectionBuffer(5x),formulatedformaximaltransfectionefficiency,8.0mLat5xconcentratedstocksolutiontomake40mLofworkingsolution
Applications:
-siRNAmiRNAmimicsormRNAtransfection
-DNA/siRNAco-transfection
-DNAoligoestransfection
Storage:
Storeat4°CforPepMuteReagentandRTforPepMuteTransfectionBuffer(5x). Ifstoredproperly,theproductisstablefor12monthsorlonger.
Advantages:
-Excellentsilencingatfinal1.0nMsiRNA
-Over95%genesilencinginawidevarietyofcells
-Onetubereactionwitheasystandardprotocol
-CompatIBLewithserumandantibiotics
-ProtocolsadaptedtoHTS
-Verylowcytotoxicity
-Veryaffordable
SilencingEfficacyComparisonbetweenPepMutesiRNATransfectionReagentandLeADIngProducts
Figure2.ExcellentsilencingofendogenouslyexpressedKIF11(alsoknownasEG5)inHEK293cellswith1.0µlofPepMutereagentand0.5pmolEG5siRNAperwellof24-wellplate.KIF11(alsoknownasEG5)encodesamotorproteinthatbelongstothekinesin-likeproteinfamilyinvolvedinchromosomepositioningandbipolarspindleformationduringcellmitosis.AreductioninKIF11levelscausesmitoticarrest.PepMutereagenteffectivelydeliversEG5siRNA(final1.0nM)toHEK293cells,leadingtomorethan80%of"round-up"phenotypeofHEK293cells48hposttransfectionovernegativecontrol(final1.0nMwithshamEG5siRNA)whileleadingsiRNAtransfectinreagents,LipofectamineRNAiMAX(RNAiMAX,1.0nMEG5siRNA)/INTERFERin(1.0nMEG5siRNA)/Dharmafect(10.0nMEG5siRNA)andjetPRIME(20nMEG5siRNA)giveaverage37%,23%,53%and48%ball-shapedphenotyperespectivelyonHEK293cells.Thephenotypeof"rounded-up"293cellswerevisualized(upperpanel)andquantified(lowerpanel)48hposttransfectionwithaNikonmicroscope.
Figure3.SilencingefficiencycomparisonofPepMuteTransfectionReagent(upperpanel)withDharmafect4(middlepanel)andLipofectamineRNAiMAX(RNAiMAX,lowerpanel)siRNATransfectionReagentsonA549cells.siRNAtargetingrenillaluciferaseatdifferentfinalconcentrationsrangingfrom0.5to20nMwasco-transfectedwithrenillaluciferasegene(0.5µgofpRL-CMVDNAperwell)bytheabovethreetransfectionreagentspermanufacturers"protocolsintoA549cellsgrowingona24-wellplate.Renillaluciferaseactivitywasdetermined36hafterpostco-transfectionwithrenillaluciferasedeterminationsystem(Promega).Theluminescencewasmeasuredfrom5.0µloflysateduring10sintegrationwithaluminometer(BeckmanCoulterLD400).Luciferaseactivitywasexpressedaslightunitsintegratedover10s(RLU)andnormalizedpermgofcellproteinbyusingtheBCAassay.Theerrorsbarsrepresentstandarddeviationderivedfromtriplicateexperiments.Luciferase-silencingefficiencywascalculatedrelativetountreatedcells.WhilePepMuteandDharmafect4reagentsdeliveredsignificantgenesilencingfrom1.0nMofrenillaluciferasesiRNA,LipofectamineRNAiMAXgavegoodknockdownonlyafter20nMwhileenhancedgeneexpressionatlowconcentrationofsiRNA(0.5and1.0nMrespectively)wasobserved.
Figure4.PepMuteTransfectionReagentknockeddownstableGFPexpressioninMCF7cell(upperpanel)andU2OScell(lowerpanel)byreverselytransfecting5.0and1.0nMGFPsiRNArespectively.Greenfluorescenceprotein(GFP)wasstablyexpressedinMCF7andU2OScells.siRNAtargetingGFPgene(rightpanel)andashamsiRNA(leftpanel)wereintroducedintoMCF7andU2OScellswithfinal5.0and1.0nMrespectivelybyreversetransfectionwithPepMuteTransfectionReagent.GFPgenesilencingwasmonitored48hposttransfectionbyaNikonfluorescencemicroscope.QuantitativeanalysisshowedthatGFPsiRNAat5.0and1.0nMdeliveredbyPepMutesiRNATransfectionReagentknockeddown90%and95%stablyexpressedGFPinMCF7andU2OScellsrespectively.
DataSheet&Protocol
-AStandardsiRNATransfectionProtocol
-AStandardsiRNA/DNACo-transfectionProtocol
-AReversesiRNATransfectionProtocolforHTS
Torequestafreetrialsample,pleaseCreateAnAccountwithustoenteryourshippingaddressandemailusatorder@Signagen.com
Testimonials:
IhadtriedyourproductpepMutetransfectionreagentonprimaryretinalneuronsandwassatisfiedwiththetransfectionefficiency.Willorder!
IreallyappreciateyousendingmeasampleofPepMutesiRNAreagent.ItestedDNA/siRNAco-transfectionusing293Tcellsandtheresultswerecompletelysatisfactory.Iwasabletoget95%knockdownofmytargetgeneat1.0nMsiRNAaswellasexpressionofplasmidDNAusingtherecommendedprotocol.Iwillhavetotestandseeifothercelllinesarealsoaseffective.IwilldefinitelythinkaboutmovingtousethisreagentoverDharmafectorOligofectamine.
ItriedPepMutereagentandIlikeit.Ithassohighefficiencyandnocytotoxicity.Iamgoingtouseit.Thankforintroducingittoourlab.
Tried3differentsiRNAswith100%silencingonA2780cell.Thatisamazing!Ialreadyplacedanorder.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
其次要看下你选择单位的规模如何，做的比较好的，还是上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
不建议你用MDCK细胞，非常难转染。
GFP发出绿色荧光的原理是Ca离子进入GFP的beta-barrel结构中引起的特定能级，因此只要这个结构仍然保持着，就可以发出荧光。
由于GFP的beta-barrel结构非常稳定，一些版本的GFP蛋白（如EGFP）甚至能抵抗94C的高温几分钟而不完全变性，因此想在溶液状态下去掉GFP的荧光是很难的，一般需要用光漂白法。
基于其非常稳定的结构，即便细胞被固定了，仍然会有一部分的GFP蛋白保持其构象而发出荧光。此时荧光可能较弱。在荧光显微镜下是有可能看得到的。
我刚开始做转染，悬浮细胞，分别做过表达和敲减，看了很多文献，大都没有提及转染后是用转染的这同一批细胞同时做pcr，wb，cck8，凋亡，细胞周期；还是说这次转染只做pcr或wb，再转染一次做cck8或细胞周期。剩下的功能试验均同前，转染一次做一次？我养的是悬浮细胞，转染后做cck8这些功能试验前需要离心换液吗？跪谢解答！
可以用移液器将细胞吸出来并高速离心，沉淀重悬于PBS中洗涤，接着就可以裂解提取蛋白了。可以用超声，酶解等等，裂解后离心收集上清。
其次要看下你选择单位的规模如何，上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
实验室一直都是用日常型质粒抽提试剂盒，转染细胞没问题。
我觉得只要是注意以下2点就可以了：
1，注意大肠杆菌(Escherichia coli)本身的污染，收集菌体沉淀时防止菌液散落，经常用75%的乙醇擦拭手套。
2，最后洗脱时最好使用无内毒素的水，我们是用注射用水的。
无论是小提还是大提我们都是用的日常型的，并没有刻意用转染级的，因为转染量大，去内毒素的操作太麻烦，损失太大。
转染分2种，一种是瞬时转染，即转染后让细胞表达目的蛋白后即提取蛋白，提一次蛋白，转染一次，这种方式一般不传代；
另一种转染为稳定转染，转染后加入一定选择压力进行筛选，没有转染的细胞不能存活，只留下转染的细胞，这种情况下可以筛选单个转染细胞，构建稳定表达某一特定蛋白或基因的细胞系。
