ProductDescription
RatCol®TypeIAcidSolubleRatTailCollagencontains100mgataconcentrationofapproximately4mg/mLina0.02Maceticacidsolution(pH2to3).RatCol®collagenissolubletelo-collagen.Eachproductincludesabottlecontaining100mgofcollagensolutionaccompaniedwithabottleofpre-formulatedneutralizingsolutionfortheformationofacollagengel.Thiscollagenproductisprovidedinuser-friendlypackagingforuseandstorage.Thisproductissterilefilteredandissuppliedasareadytousesolution.
Thisproductisidealforcoatingofsurfaces,providingpreparationofthinlayersforculturingcells,oruseasasolidgel.RatCol®collagenissuitableforapplicationsusingavarietyofcelllinesincludinghepatocytes,fibroblastsandepithelialcells.
| Parameter,Testing,andMethod | RatCol®TypeICollagen#5153 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Acid-telocollagen |
| Form | Solution |
| PackageSize | 100mg(~25mL)Kit |
| StorageTemperatureofCollagen | 2-10°C |
| StorageTemperatureofNeutralizationSolution | RoomTemperature |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 3.5-4.5mg/mL |
CollagenPurity-SilverStaining | >99% |
| pH | 3.0-3.8 |
| KineticGelTest(Minutes) | <40 |
GelFormationTubeTest(Minutes) | <40 |
ElectrophoreticPattern-CoomassieBlue | Characteristic |
| Sterility-USPmodified | Nogrowth |
| Endotoxin-LAL | <10.0EU/mL |
| Osmolality(mOsmoH2O/kg) | <35 |
| CellAttachmentAssay | Pass |
| Source | RatTailTendon |
| HydrogelYoung"sModulusE(Pa) | Characteristic |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
CoatingProcedure
Note:Employasepticpracticestomaintainthesterilityoftheproductthroughoutthepreparationandhandlingofthecollagenandothersolutions.
- Transferdesiredvolumeofcollagensolutionfromthebottletoadilutionvesselifrequired.Furtherdilutetodesiredconcentrationusingsterile0.1%aceticacidsolution.Atypicalworkingconcentrationmayrangefrom50to100ug/mL.Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
- AddappropriateamountofdilutedRatTailcollagentotheculturesurface.
- Incubateatroomtemperature,covered,for1-2hours.Aspirateanyremainingmaterial.Alternatively,incubateatroomtemperatureuntilsurfaceisdry.
- RinsecoatedsurfacescarefullywithsterilemediumorPBS,avoidscratchingsurfaces.
- Coatedsurfacesarereadyforuse.Theymayalsobestoredat2-8°Cdamporairdriedifsterilityismaintained.
3-DGelPreparationProcedureUsingtheSuppliedNeutralizationSolution
Note:Employasepticpracticestomaintainthesterilityoftheproductthroughoutthepreparationandhandlingofthecollagenandothersolutions.
Note:Itisrecommendedthatthecollagenandotherworkingsolutionsbechilledandkeptoniceduringthepreparationofthecollagen.
- Determinethedesiredvolumeofcollagenrequired.
- Transfer1partofchilledneutralizationsolutionintoasterilemixingvesselortube.
- Transfer9partsoftheRatTailCollagenintothesterilemixingvesselortubeforatotalof10parts.
- Gentlyagitatethemixtureorpipetupanddowntomix.Vortexingisnotrecommended.
- DispensetheRatTailcollagenmixtureinthedesiredsterileplatesorculturevessels.
- Incubateat37°Cfor1hourforgelformation.
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
ReferencesforRatCol®:
Dollinger,BryanR.,etal."Reactiveoxygenspeciesshieldinghydrogelforthedeliveryofadherentandnonadherenttherapeuticcelltypes."TissueEngineeringPartA23.19-20(2017):1120-1131.
Jia,Hong,etal."Thetumorcell‐secretedmatricellularproteinWISP1drivespro‐metastaticcollagenlinearization."TheEMBOJournal(2019).
PérezCardona,DavidJosé."Phenotypeanalysisofdermal-epidermalorganotypicsmadewithhacatcelllineseedingontopofthreehumanfibroblastpopulatedmatrices(polyethyleneterephthalate,fibrinorcollagenI+III)."(2017).
Karki,SuryaB.,etal."Investigationofnon-thermalplasmaeffectsonlungcancercellswithin3Dcollagenmatrices."JournalofPhysicsD:AppliedPhysics50.31(2017):315401.
Keating,M.,etal."Spatialdistributionsofpericellularstiffnessinnaturalextracellularmatricesaredependentoncell-mediatedproteolysisandcontractility."ActaBiomaterialia57(2017):304-312.
Blum,KevinM.,etal."Acellularandcellularhigh-density,collagen-fibrilconstructswithsuprafibrillarorganization."Biomaterialsscience4.4(2016):711-723.
Kaufman,Gili,andDragoSkrtic."Spatialdevelopmentofgingivalfibroblastsanddentalpulpcells:Effectofextracellularmatrix."TissueandCell49.3(2017):401-409.
ProductCertificateofAnalysis
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SafetyandDocumentation
SafetyDataSheet
CertificateofOrigin
ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
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