ProductDescription
AdvancedBiomatrixoffersPhotoGel®RUTKit,apurifiedmethacrylatedgelatinkitforvisIBLelight(400-450nm)photocrosslinkablehydrogels.PhotoGel®provides3Dtunablegelswiththeuniqueattributestobepreparedatvariousconcentrationsandcrosslinkedtoprovidevariousgelstiffness.ThisPhotoGel®kitconsistsofpurifiedmethacrylatedporcinegelatinandaVisibleLightPhotoinitiator(400-450nm).ThegelatinistypeA300bloom.
Table1:
Item | CatalogNumber | PackageSize | StorageTemperature |
MethacrylatedGelatin | #5208 | 1gram(2x500mg) | -20°C |
RutheniumPhotoinitiator | #5246 | 100mg | RoomTemperature |
SodiumPersulfatePhotoinitiator | #5247 | 500mg | RoomTemperature |
PhotoGel®isproducedfrommethacrylatedgelatinwherethegelatinhasbeenmodifiedbyreactingthefreeamines,primarilytheε-aminesgroupsofthelysineresiduesaswellasthea-aminesgroupsontheN-termini.>75%ofthetotallysineresiduesofthegelatinmoleculehavebeenmethacrylated.ThephotoinitiatorconsistsofRutheniumandSodiumPersulfatetobeformulatedin1XcellculturemediaorPBS,whichallowsvisiblelightphotocrosslinkingofthecollagenat400-450nm.
Forasepticwork,werecommendfilteringthesolubilizedrutheniumandsodiumpersulfatethrough0.2micronbuttonfilters,asthephotoinitiatorsdonotcomesterile.
Parameter,Testing,andMethod | MethacrylatedGelatin#5208 |
SterilizationMethod | Filtration |
Sterility-USPmodified | Nogrowth |
Form | LyophilizedPowder |
PackageSize | 1gram(2x500mg) |
StorageTemperature | -20°C |
ShelfLife | Minimumof6monthsfromdateofreceipt |
DegreeofMethacrylation | >75% |
GelatinSource | TypeA,300Bloom,Porcine |
HydrogelYoung"sModulusE(Pa) | Characteristic |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
3DHydrogelPreparation:
Note:Employasepticpracticestomaintainthesterilityoftheproductthroughoutthepreparationandhandlingofthecollagenandothersolutions.
Note:Thefollowinginstructionsarefora10%gelatinmethacrylatesolution.Recommendedconcentrationsare5-20%.
- Warm10mLofwarm,sterile1XPBSor1Xcellculturemediato>60°C.
- Addthe10mL’sofwarmedsolutiontotheambervialcontaining1gramoflyophilizedgelatinmethacrylate.
- Mixonashakertableorrotatorplateuntilfullysolubilized.Keepwarm(>37°C)ifpossible(eg.placeyourrotatorinanincubator)tohelpwithfullsolubilization.
- Calculatethevolumeofphotoinitiatortoaddbymultiplyingthevolumeofsolubilizedgelatinby0.02.Iftheresultingnumberis200ul,forexample,youwilladd200ulofrutheniumand200ulofsodiumpersulfate.
- Solubilizetherequiredamountofruthenium(perstep4)ataconcentrationof37.4mg/mlin1XPBSorcellculturemedia.
- Solubilizetherequiredamountofsodiumpersulfate(perstep4)ataconcentrationof119mg/mlin1XPBSorcellculturemedia.
- Addtherutheniumtothegelatinsolutionandfullymixuntilsolutionishomogeneous.
- Addthesodiumpersulfatetothegelatin/rutheniumsolutionandmixuntilsolutionishomogeneous.
- Addyourcellstothegelatin/photoinitiatorsolution.
- Dispenseyourgelatin/photoinitiator/cellsolutionintothedesireddish(ie.6-wellplate,48-wellplate).
- Forphotocrosslinking,placeprintedstructuredirectlyundera400-450nmvisiblelightcrosslinkingsource.
Longerexposureallowsmorecrosslinking,thougheachcelltypewithstandsdifferentdegreesoflightandfreeradicals(generatedbythephotoinitiator)thatmediatecrosslinking.
Anyexcessmaterialcanberefrigeratedandstored.Thematerialwillgel.Warmbackupto>30°Cforittobecomeliquidagain.Werecommendonlyaddingphotoinitiatortotheamountofgelatintobeusedatthattime.
ProductApplications
PhotoGel®Gelatinmethacrylatecanbeusedtoformcross-linkedhydrogelsfortissueengineering[1]and3Dprinting.Thecommonformsof3DprintingusingLifeink®300includeextrusion[2][3][4],inkjet[5]andphotolithography[6]).
PhotoGel®hasbeenusedforendothelialcellmorphogenesis,[7]cardiomyocytes,[8]epidermaltissue[9],injectabletissueconstructs[10],bonedifferentiation[11],andcartilageregeneration[12].
Gelatinmethacrylatehasbeenexploredindrugdeliveryapplicationsintheformofmicrospheres[13]andhydrogels[14].
References:
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878615/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040163/
- https://www.ncbi.nlm.nih.gov/pubmed/24112804
- https://www.ncbi.nlm.nih.gov/pubmed/20387987
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380738/
- https://www.ncbi.nlm.nih.gov/pubmed/28349897/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643201/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551408/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608855/
- http://pubs.acs.org/doi/abs/10.1021/bm401533y
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252258/
- https://www.ncbi.nlm.nih.gov/pubmed/24590160
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293288/
- http://www.ijpsonline.com/articles/preparation-and-characterization-of-gelatinpolymethacrylic-acid-interpenetrating-polymeric-network-hydrogels-as-a-phsensitive-deli.html
ProductReferences
ReferencesforPhotoGel®:
Rothrauff,BenjaminB.,etal."EfficacyofThermoresponsive,photocrosslinkablehydrogelsderivedfromdecellularizedtendonandcartilageextracellularmatrixforcartilagetissueengineering."Journaloftissueengineeringandregenerativemedicine12.1(2018):e159-e170.
Rothrauff,BenjaminB.,etal."Anatomicalregion-dependentenhancementof3-dimensionalchondrogenicdifferentiationofhumanmesenchymalstemcellsbysolublemeniscusextracellularmatrix."Actabiomaterialia49(2017):140-151.
Rothrauff,BenjaminB.,GuangYang,andRockyS.Tuan."Tissue-specificbioactivityofsolubletendon-derivedandcartilage-derivedextracellularmatricesonadultmesenchymalstemcells."Stemcellresearch&therapy8.1(2017):133.
Bridge,JackChristopher,etal."Electrospungelatin-basedscaffoldsasanovel3Dplatformtostudythefunctionofcontractilesmoothmusclecellsinvitro."BiomedicalPhysics&EngineeringExpress4.4(2018):045039.
Capella-Monsonís,Héctor,etal."Scaffoldsfortendontissueengineering."HandbookofTissueEngineeringScaffolds:VolumeOne.WoodheadPublishing,2019.259-298.
ProductCertificateofAnalysis
SafetyandDocumentation
SafetyDataSheet
CertificateofOrigin
ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
美国AdvancedBioMatrix(简称ABM) www.advancedbiomatrix.comAdvancedBioMatrix(简称ABM)是美国一家著名的生物公司,获得了AllerganInc的授权(Allergan用25年时间不断完善胶原蛋白相关的产品的生产工艺),将Allergan的专业和技术用于蛋白生产与检测,致力于为组织工程、细胞分析及细胞增殖等研究领域提供优质稳定的产品。AdvancedBioMatrix不断丰富已有产品线,目前可为三维细胞培养提供各种胶原蛋白、纤连蛋白、玻连蛋白、水性凝胶、不同粘度与分子量的透明质酸以及低代成纤维细胞等。在美国全部产品授权Sigma销售。AdvancedBioMatrix是组织培养,细胞分析和细胞增殖三维(3D)应用的生命科学领域的领导者。我们的产品被公认为纯度,功能性和一致性的标准。我们在生产,分离,纯化,冷冻干燥,细胞培养和蛋白质测试,粘附肽,附着因子,底物刚性和其他3D矩阵产品方面拥有丰富的专业知识。我们的专业技术和知识正在被用来确保我们的产品质量最高,批次之间一致且易于为我们的研究客户使用。
美国AdvancedBioMatrix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。AdvancedBioMatrix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3Dmatrix 产品开发方面有着丰富的经验。AdvancedBioMatrix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AdvancedBioMatrix3DMatrices 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,AdvancedBioMatrix还是唯一的预制胶原蛋白(PureColEZGel)供应商;
以下产品为AdvancedBioMatrix全球畅销品:1.PureCol 牛源I型胶原蛋白 3mg/ml#5005-100ML2.Nutragen牛源I型胶原蛋白 6mg/ml#5010-50ML3.FibriCol 牛源I型胶原蛋白 10mg/ml#5133-20ML4.VitroCol 人源I型胶原蛋白 #5007-20ML5. 弹性蛋白原 #5052-1MG6.ECMSelectArraykitUltra-36#5170-1EA7.CytoSoft(刚性可变的基底,AdvancedBioMatrix最新添加产品5190-7EA)8. 人III型胶原蛋白 #5021-10MG9. 人IV型胶原蛋白 #5022-5MG10.SilkFibroin溶液 #5154-20ML11.Fibronectin#5080-5MG12.Vitronectin#5051-0.1MG
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1、如果是提取的总蛋白,然后做WB,用β-actin或者GAPDH做内参肯定是没有问题的,这是公认的东西。
2、如果用膜蛋白提取试剂盒提取蛋白,再用β-actin作为内参似乎不妥,因为理论上来讲β-actin在膜上是不表达的。WB能做出β-actin来是因为膜蛋白提取时把胞质蛋白也提出来了。然而,如果我们试验目的是用药物处理细胞,比较处理前后某种膜蛋白的表达情况,此时用膜蛋白提取试剂盒提取膜蛋白后再用β-actin做内参似乎就不妥了,因为你根本不知道药物处理前后混杂了多少的胞质蛋白进来。如果没有胞质蛋白混进去的话,β-actin就是检测不到的。
转膜后就要区分与胶接触的一面与另一面了。话说楼主好奇的话可以跑蛋白的试试看哈。比如目的蛋白的胶用光滑面贴着胶,内参蛋白用粗糙面贴着胶一起转膜试试转膜结果看看呢。
考马斯亮兰染液也可以重复用。新配的染液10分钟即可,重复3次后要染30分钟。
一抗二抗可以重复利用,但是注意要在5%milk中加入0.2% sodium azide ,并且用完以后放入4度保存,我的经验重复使用4-5次是肯定没有问题的.如果保存不当,就会污染微生物,只能丢弃.
取名尿素。尿素含氮46%,是固体氮肥中含氮量最高的。尿素在酸、碱、酶作用下(酸、碱需加热)能水解生成氨和二氧化碳。希望我能帮助你解疑释惑。
之前是担心所需组织量不够(要求是200-300mg,我的组织只有20mg左右)才跑不出来,后来换用大鼠心脏测试,组织量绝对够。。。而且在跑western之前进行了说明书上的蛋白浓缩步骤,可是在加loADIngbuffer之后,蛋白沉淀怎么都溶解不掉~说明书也注明了如果有沉淀可以取上清继续上样,可是每次上样感觉样品往上浮,只有部分样品往孔里沉,而且跑出来什么都没有,连内参都没有。用的是β-actin的内参。。
western之前浓缩步骤如下:
1、取所得提取物,每100ul膜蛋白加入约300ul的溶解buffer和约100ul三氯乙酸(TCA)试剂,混匀后置冰上20-30min后,13000rpm,离心15min,尽可能去除上清。
2、沉淀加入1ml丙酮,室温静置10min后,13000rpm离心15min。
3、弃上清,沉淀真空旋干或置冰上干燥10min(敞开离心管盖),按适当体积比加入loadingbuffer(使用前没100ulloadingbuffer加入2-5ulβ-巯基乙醇)溶解,彻底分散(枪头反复吹吸或剧烈涡旋),煮沸5min。【注:加入loadingbuffer后如有部分难容物,可取上清继续上样。】
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