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Nutragen®collagenisknownasthestandardofallcollagensforpurity(>99%collagencontent),functionality,andthemostnative-likecollagenavailable.Nutragen®isisolatedfrombovinehidessourcedfromtheonlycontrolled,closedherdintheUnitedStates.AdvancedBiomatrix’smanufacturingprocessescomplywithstringentqualitystandardsthathaveproventoyieldunsurpassedlot-to-lotconsistency.
Nutragen®collagenisapproximately97%TypeIcollagenwiththeremainderbeingcomprisedofTypeIIIcollagen.Nutragen®collagenissuppliedatapproximatelya6mg/mLconcentrationtoprovideanextremelyfirmgel.TheconcentrationforeachspecificlotisprovidedonaCertificateofAnalysisthatisavailablewiththepurchaseofeachproduct.Nutragen®issolubleatelo-collagenin0.01NHCI,therefore,thepHisapproximately2.0.
Nutragen®collagenisidealforcoatingofsurfaces,providingpreparationofthinlayersforculturingcells,oruseasasolidgel.Nutragen®collagenisprovidedina50mLvolumeandiscontainedinuser-friendlypackagingforuseandstorage.Nutragen®issterilefilteredandissuppliedasareadytousesolution.
| Parameter,Testing,andMethod | Nutragen®TypeICollagen#5010 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Enzyme-atelocollagen |
| Form | Solution |
| PackageSize | 50mL,1000mL |
| StorageTemperature | 2-10°C |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 5.8-6.8mg/mL |
CollagenPurity-SilverStaining | >99% |
| pH | 1.9-2.1 |
| KineticGelTest(Minutes) | <40 |
| GelFormationTubeTest(Minutes) | <40 |
Fibrillogenesis(AbsorbanceUnits) | >0.5 |
ElectrophoreticPattern-CoomassieBlue | Characteristic |
| Sterility-USPmodified | Nogrowth |
| Endotoxin-LAL | <1.0EU/mL |
| Osmolality(mOsmoH2O/kg) | <35 |
| CellAttachmentAssay | Pass |
| Source | BovineHide |
| HydrogelYoung"sModulusE(Pa) | Characteristic |
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CoatingProcedure
Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
3-DGelPreparationProcedure
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.
ReferencesforNutragen®:
Tanic,J.,etal."AdseverinmodulatesmorphologyandinvasivefunctionofMCF7cells."BiochimicaetBiophysicaActa(BBA)-MolecularBasisofDisease(2019).
Rosell-García,Tamara,etal."DifferentialcleavageoflysyloxidasebythemetalloproteinasesBMP1andADAMTS2/14regulatescollagenbindingthroughatyrosinesulfatedomain."JournalofBIOLOGicalChemistry(2019):jbc-RA119.
Berger,AnthonyJ.DecouplingthePropertiesofthePhysicalMicroenvironment:TheDesignandApplicationofaGelatin-Methacrylate/CollagenCompositeHydrogel.Diss.TheUniversityofWisconsin-Madison,2018.
Berger,AnthonyJ.DecouplingthePropertiesofthePhysicalMicroenvironment:TheDesignandApplicationofaGelatin-Methacrylate/CollagenCompositeHydrogel.Diss.TheUniversityofWisconsin-Madison,2018.
Renkawitz,Jörg,etal."Nuclearpositioningfacilitatesamoeboidmigrationalongthepathofleastresistance."Nature568.7753(2019):546.
Szulc,DanielAndrzej,andHai‐LingMargaretCheng."One‐StepLabelingofCollagenHydrogelswithPolydopamineandManganesePorphyrinforNon‐InvasiveScaffoldTrackingonMagneticResonanceImaging."Macromolecularbioscience(2019):1800330.
Formica,FlorianA.,GoncaloBarreto,andMarcyZenobi-Wong."Cartilage-targetingdexamethasoneprodrugsincreasetheefficacyofdexamethasone."JournalofControlledRelease295(2019):118-129.
Dubois,Fatéméh,etal."AroleforRASSF1AintunnelingnanotubeformationbetweencellsthroughGEFH1/Rab11pathwaycontrol."CellCommunicationandSignaling16.1(2018):66.
Čermák,Vladimír,etal."RNA-seqofmacrophagesofamoeboidormesenchymalmigratoryphenotypeduetospecificstructureofenvironment."Scientificdata5(2018):180198.
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SafetyDataSheet
CertificateofOrigin
DeclarationofMaterialSource
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
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