ProductDescriptionNutragen®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,andMethodNutragen®TypeICollagen#5010SterilizationMethodFiltrationExtractionMethodEnzyme-atelocollagenFormSolutionPackageSize50mL,1000mLStorageTemperature2-10°CShelfLifeMinimumof6monthsfromdateofreceiptCollagenConcentration-Biuret5.8-6.8mg/mLCollagenPurity-SilverStaining>99%pH1.9-2.1KineticGelTest(Minutes)<40GelFormationTubeTest(Minutes)<40Fibrillogenesis(AbsorbanceUnits)>0.5ElectrophoreticPattern-CoomassieBlueCharacteristicSterility-USPmodifiedNogrowthEndotoxin-LAL<1.0EU/mLOsmolality(mOsmoH2O/kg)<35CellAttachmentAssayPassSourceBovineHideHydrogelYoung"sModulusE(Pa)CharacteristicDirectionsforUseDownloadthefullPDFversionorcontinuereADIngbelow:CoatingProcedureNote:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.Removerequiredquantityofcollagenfromthebottleanddispenseintoadilutionvessel.DiluteNutragen®inwaterto~50to100µg/ml(~1:30).A0.01NHClsolutionmayalsobeused.Swirlcontentsgentlyuntilmaterialiscompletelymixed.AddappropriateamountofdilutedNutragen®materialtotheculturesurfaceensuringthattheentiresurfaceiscoated.Incubateatroomtemperature,covered,for1-2hours.Aspirateanyremainingmaterial.Alternatively,incubateatroomtemperatureuntilsurfaceisdry.Afterincubation,aspirateanyremainingmaterial.RinsecoatedsurfacescarefullywithsterilemediumorPBS,avoidscratchingsurfaces.Coatedsurfacesarereadyforuse.Theymayalsobestoredat2-8°Cdamporairdriedifsterilityismaintained.3-DGelPreparationProcedureSlowlyadd1partofchilled10XPBSor10Xculturemediato8partsofchilledcollagensolutionwithgentleswirling.AdjustpHofmixtureto7.0–7.5.Useofsterile0.1MNaOHisrecommended.MonitorpHadjustmentcarefully(pHmeter,phenolred,orpHpaper).Adjustfinalvolumetoatotalof10partswithsterilewater.Topreventgelation,maintaintemperatureofmixtureat2-10°C.Toformgel,warmto37°C.Forbestresultsallowapproximately40minutesforgelformation.ProductQ&AIsthereDNAinthecollagenproducts?WecompletedastudytoshowthatDNAiscompletelydestroyedatpH2,anddemonstratedthatourcollagenproductsdonotcontainDNA.HowistheAminoAcidAnalysisperformed?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.Canyoudilutecollagenwith1XPBSfor2Dcoatings?Dilutingwith1XPBS(ratherthanwateror0.01NHCl)wouldhaveaneffectforcoatingpurposes.ItwouldchangethepHofthedilutedcollagensolutionfromacidtoneutralpH.ThepHchangewilltransformthecollagenmoleculesfromamolecularformtoafibrillarform;andthenthenatureofcoatingsurfacewillbechangedfromamonomericcoatingtoafibrillarcoating.WhatantibodiesdoyourecommendforyourTypeIcollagenproducts?WeusethefollowingantibodiesfromSouthernBiotech:1.1310-02–GoatAnti-TypeICollagen-FITC2.1310-08–GoatAnti-TypeICollagen-BIOT3.7100-05–Streptavidin-HRPWhatisthethicknessofthe‘molecularcollagen’attachingtothepolystyrenesurfaceofcultureware?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.Whatisthechargeofyourcollagenproducts?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.Whatisthemonomertooligomerratioinyourcollagenproducts?Usingrotaryshadowingtechniqueundertransmissionelectronmicroscopy,itwasfoundthatourcollagen,onaverage,consistsofapproximately80%monomers,13%dimers,trimers,andoligomerswiththeremaining7%collagenfragments.IspepsintreatedcollagenstillMMP-Sensitive?Yes.ThecollagenmoleculeinPureCol,Nutragen,VitroCol,andallofourotherAtelocollagenproductswerepreparedfromnativecollagenmatrixbypepsintreatmentundercontrolledconditionstoremovethenon-helicalportion,telo-peptides,onlyandthehelicalportionisintact.Inthiscase,theenzymaticactivesitesforMMP(MatrixMetalloproteinase),suchasforMammalianCollagenaseMatrixMetalloproteinase8(MMP-8),onthemoleculewaspreserved.Thesepepsintreatedcollagenproductsshouldbehaveasnativeintactcollagen.IsthereTGFBetainthecollagenproducts?TGFbetawouldhavebeendigestedwiththepepsinenzymaticdigestionstep.ItwasundetectablebySDSPAGEsilverstainaswell.Wedidn’tdoanyspecificmeasurementsbyELISAhoweverbutpresencesofTGFbetaisnotanticipated.Howdoyoudetermineproteinconcentration?WeprimarilyusetheBiuretmethod,butwealsouseBCA,AAA,andhydroxyl-prolineassays.IsmycollagenOKifitwasfrozen,orleftoutatroomtemperature?-Collagensolutionsthatarefrozentendtohaveissuesforming3Dhydrogels,andwilllikelynotwork.Thesolutionsshouldstillbegoodfor2Dcoatings.-Collagensolutionsthatareleftoutatroomtemperatureforextendedperiodsoftimemayshowsignsofdegradation,whichwillaffecttheformationof3Dhydrogels.Itislikelystillfinefor2Dcoatings.Ourrecommendationisthis:Ifyouareusingtheproductdirectlyforapublication,wehighlysuggestbuyinganewbottleiftheoneyouhavewascompromised.ProductReferencesReferencesforNutragen®: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.ProductCertificateofAnalysisNoresultfor.ProductVideosHowtoMake3DCollagenHydrogelsVideoSeeMore>TelovsAteloCollagenVideoSeeMore>SeedingCollagenGelswithCellsVideoSeeMore>4KeyComponentsfor3DCollagenGelsVideoSeeMore>CollagenConcentrationvsGelStiffnessVideoSeeMore>CoatingPlateswithCollagenVideoSeeMore>CoatingaGlassCoverslipwithCollagenVideoSeeMore>SeeMoreSafetyandDocumentationSafetyDataSheetCertificateofOriginDeclarationofMaterialSourceProductDisclaimerThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.

美国Advanced BioMatrix是3D组织培养、细胞检测和细胞增殖等领域实验解决方案的佼佼者。Advanced BioMatrix在分离、纯化、冻干、细胞培养和蛋白检测、多肽粘附、附着因子、基质硬度和其他3D matrix 产品开发方面有着丰富的经验。Advanced BioMatrix的研发经验和专业知识确保其产品可达到最佳质量，并保证产品之间一致性，方便研究客户使用。

以下为Advanced BioMatrix 3D Matrices 产品竞争优势：
1. 提供高纯度和成分确定的胞外基质；
2. 超过1000余篇文献引用PureCol产品，品质非常均一；
3. 在3D培养基领域可提供最全面的产品线；
4. 唯一可提供特异性刚性有机硅基板的公司（CytoSoft）;
5. 唯一可提供可溶性丝纤蛋白的供应商（可运用于多种3D培养）；
6. 如果客户首次接触3D胶原凝胶，Advanced BioMatrix还是唯一的预制胶原蛋白(PureCol EZ Gel)供应商。

以下产品为Advanced BioMatrix全球畅销品：
1. PureCol 牛源I型胶原蛋白 3 mg/ml #5005-100ML
2. Nutragen牛源I型胶原蛋白 6 mg/ml #5005-100ML
3. FibriCol 牛源I型胶原蛋白 10 mg/ml #5133-20ML
4. VitroCol 人源I型胶原蛋白 #5007-20ML
5. 弹性蛋白原 #5052-1MG
6. ECM Select Array kit Ultra-36 #5170-1EA
7. CytoSoft（刚性可变的基底，Advanced BioMatrix最新添加产品5190-7EA）
8. 人III型胶原蛋白 #5021-10MG
9. 人IV型胶原蛋白 #5022-5MG
10. Silk Fibroin溶液 #5154-20ML
11. Fibronectin #5080-5MG
12. Vitronectin #5051-0.1MG

Advanced BioMatrix最畅销的产品是PureCol（5005-100ML）。目前已有上千发表的论文使用这款产品。（Nutragen 5010、fibricol 5133和PureCol的物质成分一样，仅仅是浓度不同）浓度越高，胶原蛋白硬度越强。