ProductDescription
TeloCol®-6bovinecollagensolutionisderivedfromanacidextractionprocessyieldingatelopeptide-intactcollagen.Thepro-peptideregionsatbothendsofthecollagenchain,N-andC-telopeptideregions,aremaintained.
TeloCol®-6collagenisapproximately95%TypeIcollagenwiththeremainderbeingcomprisedofTypeIIIcollagen.Eachproductincludesabottlecontaining50mlofcollagensolutionaccompaniedwithabottleofpre-formulatedneutralizingsolutionfortheformationofacollagengel.Thisproductissuppliedataconcentrationofapproximately6mg/ml.TeloCol®-6issterilefilteredandprovidedinuser-friendlypackagingforuseandstorage.
TeloCol®-6isidealforcoatingofsurfaces,providingpreparationofthinlayersforculturingcells,oruseasasolidgel.
| Parameter,Testing,andMethod | TeloCol®-6TypeICollagen#5225 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Acid-telocollagen |
| Form | Solution |
| PackageSize | 50mLKit |
| StorageTemperatureofCollagen | 2-10°C |
| StorageTemperatureofNeutralizationSolution | RoomTemperature |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 5.8-6.8mg/mL |
CollagenPurity-SilverStaining | >99% |
| pH | 1.9-3.0 |
| KineticGelTest(Minutes) | <40 |
| GelFormationTubeTest(Minutes) | <40 |
Fibrillogenesis(AbsorbanceUnits) | >0.35 |
ElectrophoreticPattern-CoomassieBlue | Characteristic |
| Sterility-USPmodified | Nogrowth |
| Endotoxin-LAL | <10.0EU/mL |
| Osmolality(mOsmoH2O/kg) | <35 |
| CellAttachmentAssay | Pass |
| Source | BovineHide |
| HydrogelYoung"sModulusE(Pa) | Characteristic |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
Note:Employasepticpracticestomaintainthesterilityoftheproductthroughoutthepreparationandhandlingofthecollagenandothersolutions.
CoatingProcedure
- TransferdesiredvolumeofTeloCol®-6collagensolutionfromthebottletoadilutionvesselifrequired.Furtherdilutetodesiredconcentrationusingsterile0.01NHClsolution.Swirlcontentsgentlyuntilmaterialiscompletelymixed.Atypicalworkingconcentrationmayrangefrom50to100ug/ml.Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
- AddappropriateamountofdilutedTeloCol®-6collagentotheculturesurfaceensuringthattheentiresurfaceiscoated.
- Incubateatroomtemperature,covered,for1-2hours.Aspirateanyremainingmaterial.Alternatively,incubateatroomtemperatureuntilsurfaceisdry.
- RinsecoatedsurfacescarefullywithsterilemediumorPBS,avoidscratchingsurfaces.
- Coatedsurfacesarereadyforuse.Theymayalsobestoredat2-8°Cdamporairdriedifsterilityismaintained.
3-DGelPreparationProcedureusingthesuppliedNeutralizationSolution
Note:Itisrecommendedthatthecollagenandotherworkingsolutionsbechilledandkeptoniceduringthepreparationofthecollagen.
- Determinethedesiredvolumeofcollagenrequired.
- Transfer1partofchilledneutralizationsolutionintoasterilemixingvesselortube.
- Transfer9partsoftheTeloCol®-6collagenintothesterilemixingvesselortubeforatotalof10parts.
- Gentlyagitatethemixtureorpipetupanddowntomix.Vortexingisnotrecommended.
- Dispensethecollagenmixtureinthedesiredsterileplatesorculturevessels.
- Incubateat37°Cfor1hourforgelformation.
3-DGelPreparationProcedurewithoutusingthesuppliedNeutralizationSolution
Note:Itisrecommendedthatthecollagenandotherworkingsolutionsbechilledandkeptoniceduringthepreparationofthecollagen.
- Slowlyadd1partofchilled10XPBSor10Xculturemediato8partsofchilledcollagensolutionwithgentleswirling.
- AdjustpHofmixtureto7.2–7.6usingsterile0.1MNaOH.MonitorpHadjustmentcarefully(pHmeter,phenolred,orpHpaper).Mixwithgentleswirling.
- Adjustfinalvolumetoatotalof10partswithsterilewaterandmix.
- Topreventgelation,maintaintemperatureofmixtureat2–10°C.
- DispensetheTeloCol®-6collagenmixtureinthedesiredsterileplatesorculturevessels.
- Toformgel,warmto37°C.Allowapproximately30to60minutesforgelformation.
ProductQ&A
WecompletedastudytoshowthatDNAiscompletelydestroyedatpH2,anddemonstratedthatourcollagenproductsdonotcontainDNA.
Thecollagenisfullyhydrolyzed.TheaminoacidanalysisisdoneusingtheWatersAccQ-Tagderivatizationmethod.Duringtheacidhydrolysisstep,asparagine(N)isconvertedtoasparticacid(D)andglutamine(Q)isconvertedtoglutamicacid(E).Tryptophan(W),ifpresent,isdestroyedduringacidhydrolysis.Experimentally,onecandeterminethepicomoles(pmol)ofeachaminoacidperinjecteddetectedusingaminoacidstandards.Fortheconcentrationdetermination,thetotalnumberofpmolofeachaminoacidissummedtogetthetotalpmolofthe18aminoacidsdetected.Thetotalpmolaminoacidsisdividedbythetheoreticalnumberofaminoacidresiduesincollagenbasedonthepublishedsequence.Theresultisthepmolofcollageninjected.Theresultisthenmultipliedbythedilutionand300,000isusedasthecollagenmolecularweighttogettomg/mL.Themolecularweightofcollagenisnotwellagreedupon.
Dilutingwith1XPBS(ratherthanwateror0.01NHCl)wouldhaveaneffectforcoatingpurposes.ItwouldchangethepHofthedilutedcollagensolutionfromacidtoneutralpH.ThepHchangewilltransformthecollagenmoleculesfromamolecularformtoafibrillarform;andthenthenatureofcoatingsurfacewillbechangedfromamonomericcoatingtoafibrillarcoating.
WeusethefollowingantibodiesfromSouthernBiotech:
1.1310-02–GoatAnti-TypeICollagen-FITC
2.1310-08–GoatAnti-TypeICollagen-BIOT
3.7100-05–Streptavidin-HRP
ThemajorcollagenmolecularspeciesinourTypeIcollagenproductsaremonomers(approx.70%),buttherearedimers,trimersandafewpercentagesofoligomerstoo(approx.30%)withsomeminoramountsofcollagenfragments.Thecollagenmonomerisarodshapedmoleculewith300nminlengthand1.5nmindiameter.Thedimer,trimerandoligomerare600nm,900nmandevenlongerinlengthrespectively.Accordingtothecoatingprocedures,thecollagenmoleculesareattachedtothechargedpolystyrenesurfacerandomlybychargeoraffinityinacidconditionsduringthe1-2hrsincubationperiodat37°C,andanyunattachedmaterialsareremovedbyaspirationandrinsing.Therefore,thecoatedsurfaceisasinglelayerofcollagenmonomer,dimer,trimerandoligomermixtures.Thethicknessofthemono-molecularlayerisdependentonhowthosemoleculesareattachedonthesurface.Thecoatingdensitythicknesswouldgenerallybecharacterizedasa1moleculethicknesswhichcouldberangingfromafewnanometerstoafewhundrednanometerswiththewholesurfacebeingcoveredbycollagen.
ThenetchargeofTypeIcollagenproducts’(PureCol®,BovineCollagenandVitroCol®,HumanCollagen)moleculeisdirectlyrelatedtothepH.AtanacidicpH,theaminoacids(zwitterions)alongthecollagenmoleculearepositivelycharged,makingtheentirecollagenmoleculepositive.Attheisoelectricpoint(orzone)ofcollagen,aroundpH7-8,theaminoacidsalongthecollagenmoleculearepositivelyandnegativelycharged,makingthenetchargeofthecollagenmoleculeclosetozero.AtabasicpH,theaminoacidsalongthecollagenmoleculewerenegativelycharged,makingtheentirecollagenmoleculenegative.
Further,thenatureofthechargeofthecollagencoatingsurfacewillbedependentonthetypeofcoatingapplied.ForamonomericcollagencoatingswhenthecollagenisappliedunderanacidicpHcondition,thesurfaceispositivelycharged.IfthesurfaceisrinsedwithpHneutralbufferormediathenitwillchangethechargeofthecollagensurfacenetchargeclosetozero.Fora3Dgelcoating,thecollagenpreparedunderneutralpH;thenetchargeofthecollagensurfaceisclosetozero.
Usingrotaryshadowingtechniqueundertransmissionelectronmicroscopy,itwasfoundthatourcollagen,onaverage,consistsofapproximately80%monomers,13%dimers,trimers,andoligomerswiththeremaining7%collagenfragments.
Yes.ThecollagenmoleculeinPureCol,Nutragen,VitroCol,andallofourotherAtelocollagenproductswerepreparedfromnativecollagenmatrixbypepsintreatmentundercontrolledconditionstoremovethenon-helicalportion,telo-peptides,onlyandthehelicalportionisintact.Inthiscase,theenzymaticactivesitesforMMP(MatrixMetalloproteinase),suchasforMammalianCollagenaseMatrixMetalloproteinase8(MMP-8),onthemoleculewaspreserved.
Thesepepsintreatedcollagenproductsshouldbehaveasnativeintactcollagen.
TGFbetawouldhavebeendigestedwiththepepsinenzymaticdigestionstep.ItwasundetectablebySDSPAGEsilverstainaswell.Wedidn’tdoanyspecificmeasurementsbyELISAhoweverbutpresencesofTGFbetaisnotanticipated.
WeprimarilyusetheBiuretmethod,butwealsouseBCA,AAA,andhydroxyl-prolineassays.
-Collagensolutionsthatarefrozentendtohaveissuesforming3Dhydrogels,andwilllikelynotwork.Thesolutionsshouldstillbegoodfor2Dcoatings.
-Collagensolutionsthatareleftoutatroomtemperatureforextendedperiodsoftimemayshowsignsofdegradation,whichwillaffecttheformationof3Dhydrogels.Itislikelystillfinefor2Dcoatings.
Ourrecommendationisthis:Ifyouareusingtheproductdirectlyforapublication,wehighlysuggestbuyinganewbottleiftheoneyouhavewascompromised.
ProductReferences
ReferencesforTeloCol®:
Drzewiecki,KathrynE.,etal.“CircularDichroismSpectroscopyofCollagenFibrillogenesis:ANewUseforanOldTechnique.”BiophysicalJournal,vol.111,no.11,2016,pp.2377–2386.,doi:10.1016/j.bpj.2016.10.023
Wingender,Brian,etal.“BiomimeticOrganizationofCollagenMatricestoTemplateBone-likeMicrostructures.”MatrixBIOLOGy,vol.52-54,2016,pp.384–396.,doi:10.1016/j.matbio.2016.02.004.
Burla,F.,Tauber,J.,Dussi,S.,Gucht,J.V.D.&Koenderink,G.H.Stressmanagementincompositebiopolymernetworks.NaturePhysics15,549–553(2019).
ProductCertificateofAnalysis
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SafetyandDocumentation
SafetyDataSheet
CertificateofOrigin
DeclarationofMaterialSource
ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
美国AdvancedBioMatrix（简称ABM） www.advancedbiomatrix.comAdvancedBioMatrix（简称ABM）是美国一家著名的生物公司，获得了AllerganInc的授权(Allergan用25年时间不断完善胶原蛋白相关的产品的生产工艺)，将Allergan的专业和技术用于蛋白生产与检测，致力于为组织工程、细胞分析及细胞增殖等研究领域提供优质稳定的产品。AdvancedBioMatrix不断丰富已有产品线，目前可为三维细胞培养提供各种胶原蛋白、纤连蛋白、玻连蛋白、水性凝胶、不同粘度与分子量的透明质酸以及低代成纤维细胞等。在美国全部产品授权Sigma销售。AdvancedBioMatrix是组织培养，细胞分析和细胞增殖三维（3D）应用的生命科学领域的领导者。我们的产品被公认为纯度，功能性和一致性的标准。我们在生产，分离，纯化，冷冻干燥，细胞培养和蛋白质测试，粘附肽，附着因子，底物刚性和其他3D矩阵产品方面拥有丰富的专业知识。我们的专业技术和知识正在被用来确保我们的产品质量最高，批次之间一致且易于为我们的研究客户使用。
美国AdvancedBioMatrix是3D组织培养、细胞检测和细胞增殖等领域实验解决方案的佼佼者。AdvancedBioMatrix在分离、纯化、冻干、细胞培养和蛋白检测、多肽粘附、附着因子、基质硬度和其他3Dmatrix 产品开发方面有着丰富的经验。AdvancedBioMatrix的研发经验和专业知识确保其产品可达到最佳质量，并保证产品之间一致性，方便研究客户使用。以下为AdvancedBioMatrix3DMatrices 产品竞争优势：1. 提供高纯度和成分确定的胞外基质；2. 超过1000余篇文献引用PureCol产品，品质非常均一；3. 在3D培养基领域可提供最全面的产品线；4. 唯一可提供特异性刚性有机硅基板的公司（CytoSoft）;5. 唯一可提供可溶性丝纤蛋白的供应商（可运用于多种3D培养）；6. 如果客户首次接触3D胶原凝胶，AdvancedBioMatrix还是唯一的预制胶原蛋白(PureColEZGel)供应商；
以下产品为AdvancedBioMatrix全球畅销品：1.PureCol 牛源I型胶原蛋白 3mg/ml#5005-100ML2.Nutragen牛源I型胶原蛋白 6mg/ml#5010-50ML3.FibriCol 牛源I型胶原蛋白 10mg/ml#5133-20ML4.VitroCol 人源I型胶原蛋白 #5007-20ML5. 弹性蛋白原 #5052-1MG6.ECMSelectArraykitUltra-36#5170-1EA7.CytoSoft（刚性可变的基底，AdvancedBioMatrix最新添加产品5190-7EA）8. 人III型胶原蛋白 #5021-10MG9. 人IV型胶原蛋白 #5022-5MG10.SilkFibroin溶液 #5154-20ML11.Fibronectin#5080-5MG12.Vitronectin#5051-0.1MG
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Bone Mesenchymal Stem Cells 作为一个细胞群体，还没有发现有特定细胞表面marker. 对于那些可以代表自我更新和分化的marker, 也不清楚到底要发现哪一个的表达才能确定该细胞就是BMSC。
目前常用的方法，就是采用培养，colony-forming unit-fibroblasts (CFU-F)这个方法。一般BMSC可以24-48小时贴壁。
流式细胞计数，比如STRO-1，但是一般认为STRO-1阳性的细胞更趋向于造血干细胞，和BMSC简单区别还不是很清楚。
这里有个培养分化的产品
http://www.rndsystems.com/pdf/SC020.pdf
GlucosestarvationcausestranslocationofAMPKβ2tothelysosomeinHEK-293cellsthatisdependentonN-myristoylation.Theexperimentwasperformedinβ2KOcellsasinFig.1c,exceptthatthelysosomalMarkerLAMP1(taggedwithRFP)wasco-expressedwiththewild-typeormutantAMPKβ2.Upperpanelsshowmergedimagesstainedblue(4′,6-diamidino-2-phenylindole(DAPI),nuclei),red(LAMP1,lysosomes)andgreen(AMPKβ2,detectedusingantibodyvalidatedine),incellsincubatedwithorwithoutglucosefor20 min.Lowersmallpanelsaremagnificationsoftheareasindicatedbydashedboxesintheupperpanels,showing(LtoR)redandgreenchannelsandmergedimages.
下面的这段话是图注，图注的意思我明白，但是我想知道merge后的图看什么颜色的荧光，蓝色是细胞核，红色是lysosome(位于胞质），绿色是AMPKβ2，该实验是想观察AMPKβ2是否转位到lysosome上了，如果确实发生了AMPKβ2转位到lysosome上，那么merge后是红色与绿色融合在一起，是吗？融合在一起发什么颜色的光了？
