ProductDescription
FlexiCol®isisolatedfromporcinehidesandisasolubleatelo-collagenin0.01NHCI.FlexiCol®collagenisapproximately97%TypeIcollagenwiththeremainderbeingcomprisedofTypeIIIcollagen.FlexiCol®collagenissuppliedatapproximatelya3mg/mlconcentration.
FlexiCol®collagenisidealforcoatingofsurfaces,providingpreparationofthinlayersforculturingcells,oruseasasolidgel.FlexiCol®collagenisprovidedinauser-friendlypackagingforuseandstorage.FlexiCol®issterilefilteredandissuppliedasareadytousesolution.
| Parameter,Testing,andMethod | FlexiCol®TypeICollagen#5169 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Enzyme-atelocollagen |
| Form | Solution |
| PackageSize | 100mL |
| StorageTemperature | 2-10°C |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 2.9-3.3mg/mL |
| pH | 1.9-2.2 |
| 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 | PorcineHide |
| HydrogelYoung"sModulusE(Pa) | Characteristic |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
CoatingProcedure
Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
- Removerequiredquantityofcollagenfromthebottleanddispenseintoadilutionvessel.
- DiluteFlexiCol®inwaterto~50to100µg/ml(~1:30).A0.01NHClsolutionmayalsobeused.
- Swirlcontentsgentlyuntilmaterialiscompletelymixed.
- AddappropriateamountofdilutedFlexiCol®materialtotheculturesurfaceensuringthattheentiresurfaceiscoated.
- Incubateatroomtemperature,covered,for1-2hours.Aspirateanyremainingmaterial.Alternatively,incubateatroomtemperatureuntilsurfaceisdry.
- RinsecoatedsurfacescarefullywithsterilemediumorPBS,avoidscratchingsurfaces.
- Coatedsurfacesarereadyforuse.Theymayalsobestoredat2-8°Cdamporairdriedifsterilityismaintained.
3-DGelPreparationProcedure
- Slowlyadd1partofchilled10XPBSor10Xculturemediato8partsofchilledcollagensolutionwithgentleswirling.
- AdjustpHofmixtureto7.0–7.5usingsterile0.1MNaOH.MonitorpHadjustmentcarefully(pHmeter,phenolred,orpHpaper).
- Adjustfinalvolumetoatotalof10partswithsterilewater.
- Topreventgelation,maintaintemperatureofmixtureat2-10°C.
- Toformgel,warmto37°C.Forbestresultsallowapproximately90to120minutesforgelformation.
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.
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ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
