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.
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