ProductDescription
PureCol®collagenisknownasthestandardofallcollagensforpurity(>99.9%collagencontent),functionality,andthemostnative-likecollagenavailable.PureCol®isisolatedfrombovinehidessourcedfromtheonlycontrolled,closedherdintheUnitedStates.AdvancedBiomatrix’smanufacturingprocessescomplywithstringentqualitystandardsthathaveproventoyieldunsurpassedlot-to-lotconsistency.
PureCol®collagenisapproximately97%TypeIatelocollagenwiththeremainderbeingcomprisedofTypeIIIcollagen.PureCol®collagenissuppliedatapproximatelya3mg/mlconcentration.TheconcentrationforeachspecificlotisprovidedonaCertificateofAnalysisthatisavailablewiththepurchaseofeachproduct.PureCol®issolubleatelocollagenin0.01NHCI,therefore,thepHis2.PureCol®collagenisidealforcoatingofsurfaces,providingpreparationofthinlayersforculturingcells,oruseasasolidgel.
PureCol®collagenisprovidedinauser-friendlypackagingforuseandstorage.PureCol®issterilefilteredandissuppliedasareadytousesolution.
| Parameter,Testing,andMethod | PureCol®TypeICollagen#5005 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Enzyme-atelocollagen |
| Form | Solution |
| PackageSize | 100mL,1000mL |
| StorageTemperature | 2-10°C |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 2.9-3.2mg/mL |
CollagenPurity-SilverStaining | >99.9% |
| 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 |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
CoatingProcedure
Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
- Removerequiredquantityofcollagenfromthebottleanddispenseintoadilutionvessel.
- DilutePureCol®inwaterto~50to100µg/ml(~1:30).A0.01NHClsolutionmayalsobeused.
- Swirlcontentsgentlyuntilmaterialiscompletelymixed.
- AddappropriateamountofdilutedPureCol®materialtotheculturesurfaceensuringthattheentiresurfaceiscoated.
- Incubateatroomtemperature,covered,for1-2hours.Aspirateanyremainingmaterial.Alternatively,incubateatroomtemperatureuntilsurfaceisdry.
- RinsecoatedsurfacescarefullywithsterilemediumorPBS,avoidscratchingsurfaces.
- Coatedsurfacesarereadyforuse.Theymayalsobestoredat2-10°Cdamporairdriedifsterilityismaintained.
3-DGelPreparationProcedure
- Slowlyadd1partofchilled10XPBSor10Xculturemediato8partsofchilledcollagensolutionwithgentleswirling.
- AdjustpHofmixtureto7.0–7.5usingsterile0.1MNaOH.MonitorpHadjustmentcarefully(pHmeter,phenolred,orpHpaper).
- Adjustfinalvolumetoatotalof10partswithsterilewater.
- Topreventgelation,maintaintemperatureofmixtureat2–10°C.
- Toformgel,warmto37°C.Allowapproximately90to120minutesforgelformation.
ProductQ&A
ThepurityofPureCol®collagenisdeterminedbySDS-PAGE,sodiumdodecylsulfatepolyacrylamidegelelectrophoresisinconjunctionwithbacterialCollagenasesensitivityandsilverstainingtechniqueswithamethodsensitivityof99.9%.ItwasfoundthatPureCol®collagenis95to98%TypeIcollagenandtheremainderbeingcomprisedofTypeIIIcollagen.
SDSpolyacrylamidegelelectrophoresisdemonstratesthepresenceofalpha,betaandgammacomponentsinanappropriateratioofapproximately40:30:30,respectively.PureCol®collagenisanativecollagenasjudgedbypolarimetryandtrypsinsensitivityalthoughtheproductdoescontainalowpercentageofcollagenfragmentsorshortenedhelices.
Conclusion:Withthetestmethodsensitivityof99.9%(SDS-PAGEgelelectrophoresisinconjunctionwithbacterialcollagenasesensitivityandsilverstainingtechniques)andnootherproteinspresentinthepreparation,itcanbeconcludedthatthepurityofthePureCol®collagenis99.9%.
TheviscosityofPureCol®is~32cp.
PureCol®producthasanisoelectriczoneinsteadofisoelectricpoint.TheisoelectriczoneispH7to8.Inaddition,thecollagenmoleculesinthePureCol®productwillcomeoutofsolutionstartingatapHabove5.5andreachitsplateauatpH7to8thengraduallytaperingoffatpH8to9.5.
Reductionofacommerciallyavailable,pepsin-solubilized,bovinedermalcollagen(Vitrogen100)(PureCol’soldproductname)withsodium[3H]borohydrideprovidedradiolabeledcollagenpreparationswithspecificactivitiesrangingfrom7.1-12.0muCi/mgcollagen.Thesespecificactivitieswere2-3timesgreaterthanthoseobtainedbyreductionofintactrattailtendoncollagenundersimilarconditions.
Thealpha,beta,andhigheraggregatecomponentsoftypeIcollagenwereradiolabeledaswellasthealphacomponentofasmallamountoftypeIIIcollagenpresentinthesamples.Fractionationofcyanogenbromidepeptidesshowedthatalpha1(I)CB7,alpha1(I)CB8,andalpha2(I)CB3,5werethepredominantpeptideslabeledbythisprocedure.AminoacidanalysisindicatedthatthemajorityoftheradioactivitywasinreducIBLecross-links,precursorsofthesecross-links,andinhexosyllysineresidues.
Reconstitutionexperimentscomparingthisradiolabeledcollagenwithnonlabeledcollagenshowedthemtobeindistinguishable.Bacterialcollagenasedigestionofthisreconstitutedfibrillarcollageninbothalightlycross-linked(glutaraldehyde0.0075%)andnoncross-linkedformprovidedevidencethatdigestionoflabeledandnonlabeledcollagensproceededatsimilarrates.Thus,labelingdidnotchangethepropertiesofthecollagen.Cross-linkingmadethepreparationrefractorytoproteolyticdegradation.Injectionoffibrillarcollagenpreparations,spikedwithradiolabeledcollagen,intotheguineapigdermisfollowedbyquantitationoftheamountofradioactivityrecoveredfromimplantsitesasafunctionoftime,indicatedthatthelightlycross-linkedsamplesalsoweremoreresistanttodegradationinvivothanthenoncross-linkedpreparation.
Thehalf-lifeofnoncross-linkedcollagenwasabout4dayswhilethatofthecross-linkedcollagenwasabout25days.Thesedegradationratesweremuchfasterthanobservedforsimilar,nonlabeledsamplesinjectedintothedermisofhumans,presumablyduetoahighermetabolicactivityintheguineapigdermis.
SincethecollageninPureColcollagencontainsapproximately95%TypeIbovinecollagenand5%TypeIIIbovinecollagen,ananti-bovinecollagenTypeIantibodyforyourstudycanbeused.
Thereisnodifference.Vitrogenwastheoldtradename,andPureCol®isthenewtradename.
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
BecausePureCol®hasbeencitedinover2000publications,wehaveonlypostedafewbelow:
Sorensen,JacobR.,etal."Analteredresponseinmacrophagephenotypefollowingdamageinagedhumanskeletalmuscle:implicationsforskeletalmusclerepair."TheFASEBJournal(2019):fj-201900519R.
Sorensen,JacobR.,etal."Analteredresponseinmacrophagephenotypefollowingdamageinagedhumanskeletalmuscle:implicationsforskeletalmusclerepair."TheFASEBJournal(2019):fj-201900519R.
Colaço,E.,etal."HierarchicalCollagen-HydroxyapatiteNanostructuresDesignedThroughLayer-by-LayerAssemblyofCrystal-DecoratedFibrils."J.,HierarchicalCollagen-HydroxyapatiteNanostructuresDesignedThroughLayer-by-LayerAssemblyofCrystal-DecoratedFibrils(May13,2019)(2019).
Schwerdtfeger,LukeA.,etal."Humancolonfunctionexvivo:Dependenceonoxygenandsensitivitytoantibiotic."PloSone14.5(2019):e0217170.
Cardoso,Ana,etal."MiR-144overexpressionasapromisingtherapeuticstrategytoovercomeglioblastomacellinvasivenessandresistancetochemotherapy."Humanmoleculargenetics(2019).
Steele,HannahE.,etal."MechanotransductionofmitochondrialAMPKanditsdistinctroleinflow-inducedbreastcancercellmigration."Biochemicalandbiophysicalresearchcommunications514.2(2019):524-529.
Gehwolf,Renate,etal."GlobalResponsesofIl-1β-Primed3DTendonConstructstoTreatmentwithPulsedElectromagneticFields."Cells8.5(2019):399.
Alexander,Frank,SebastianEggert,andDoriellePrice."Label-FreeMonitoringof3DTissueModelsviaElectricalImpedanceSpectroscopy."(2019):1-24.
Matysik-Woźniak,Anna,etal."ExaminationofKynurenineToxicityonCornealandConjunctivalEpithelium:InvitroandinvivoStudies."Ophthalmicresearch(2019):1-12.
Compton,Clayton,etal."ReconstitutionoftheVentricularEndocardiumWithinAcellularHearts."RegenerativeEngineeringandTranslationalMedicine(2019):1-11.
Müller,A.L.,etal."4.IdentificationofmiR-301ainPrimaryHumanAtrialFibroblastsandBoneMarrow-DerivedMesenchymalProgenitorCellstoAttenuateEndogenousDifferentiationintoPro-FibroticCells."DifferentiationofPrimaryHumanPro-FibroticMesenchymalCellsInfluencedbyExtracellularMatrixEnvironmentDeterminedbyMicro-RNAExpression(2018):130.
Doblinger,Nina,etal."Impactofhydroxyethylstarchandmodifiedfluidgelatinongranulocytephenotypeandfunction."Transfusion(2019).
Elisabeth,etal."Pro-InflammatoryResponsesinHumanBronchialEpithelialCellsInducedbySporesandHyphalFragmentsofCommonDampIndoorMolds."Internationaljournalofenvironmentalresearchandpublichealth16.6(2019):1085.
Dodmane,PuttappaR.,etal."BiphasicchangesinairwayepithelialcellEGFreceptorbindingandphosphorylationinducedbycomponentsofhogbarndust."Experimentallungresearch44.10(2018):443-454.
McClellan,Alyce,etal."Anovelmechanismfortheprotectionofembryonicstemcellderivedtenocytesfrominflammatorycytokineinterleukin1beta."Scientificreports9(2019).
Wang,Weiling,etal."Aquaporin-3deficiencyslowscystenlargementinexperimentalmousemodelsofautosomaldominantpolycystickidneydisease."TheFASEBJournal(2019):fj-201801338RRR.
Teo,JyeYng,etal."SurfacetetheringofstemcellswithH2O2-responsiveanti-oxidizingcolloidalparticlesforprotectionagainstoxidation-induceddeath."Biomaterials201(2019):1-15.
Gehwolf,Renate,etal."3D-EmbeddedCellCulturestoStudyTendonBIOLOGy."(2019):1-11.
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ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
