Product Description
Advanced BioMatrix offers PhotoCol®, a purified methacrylated Type I bovine collagen kit. PhotoCol® provides native-like 3D collagen gels with the unique attributes to be prepared at various concentrations and crosslinked to provide various gel stiffness.
The PhotoCol® kit consists of purified methacrylated Type I bovine collagen as the core component with other support reagents in the kit. Table 1 provides a list of the kit components.
Table 1:
| Item | Catalog Number | Package Size | Storage Temperature |
| Methacrylated Collagen | #5198 | 100 mg | 2-10°C |
| 20 mM Acetic Acid | #5079 | 50 mL | 2-30°C |
| Neutralization Solution | #5205 | 10 mL | Room Temperature |
| Irgacure Photoinitiator | #5200 | 100 mg | 2-10°C |
The methacrylated Type I collagen is produced from telo-peptide intact bovine collagen where the collagen has been modified by reacting the free amines, primarily the ε-amines groups of the lysine residues as well as the a-amines groups on theN-termini. > 20% of the total lysine residues of the collagen molecule have been methacrylated. The collagen is extracted from bovine hide and contains a high monomer content.
The collagen starting material was isolated from a closed herd and purified using controlled manufacturing processes.A bottle of 20 mM acetic acid solution is provided to solubilize the lyophilized methacrylated collagen at concentrations ranging from 3 to 8 mg/ml. The neutralization solution consists of an alkaline 10X phosphate buffered saline (PBS) solution which provides physiological salts and pH in the final mixture. The photoinitiator consists of Irgacure 2959 to be formulated in methanol (methanol not included) which allows UV crosslinking of the collagen at 365 nm.
| Parameter, Testing, and Method | Methacrylated Collagen#5198 |
| Sterilization Method | Filtration |
| Extraction Method | Acid - Telocollagen |
| Form | Lyophilized Powder |
| Degree of Methacrylation | >20% |
| Package Size | 100 mg |
| Shelf Life | Minimum of 6 months from date of receipt |
| Shelf Life After Reconstitution | 2 Months |
Collagen Purity - Silver Staining | >99% |
| Kinetic Gel Test (Minutes) | <40 |
| Gel Formation Tube Test (Minutes) | < 40 |
Electrophoretic Pattern - Coomassie Blue | Characteristic |
| Sterility - USP modified | No growth |
| Endotoxin - LAL | <10.0 EU/mL |
| Source | Bovine Hide |
| Hydrogel Young's Modulus E (Pa) | Characteristic |
Directions for Use
Download the full Directions for Use PDF
Product Applications
PhotoCol®Methacrylated Collagen is a photocrosslinkable type I collagen for 3D cell culture and bioprinting.
As the most abundant protein in the body, collagen is a key component for cell culture. Collagen methacrylate can be used as a rapidly self-assembling type I collagen to form cross-linked hydrogels for tissue engineering[1]. It has been used with mesenchymal stem cells[2], fibroblasts, adipose derived stem cells, epithelial cells, and many more.
Collagen methacrylate is useful for forming scaffolds with varying degree of stiffness, by altering collagen concentration or the dose of UV-light exposure[2]. It is also being extensively characterized for its usefulness in 3D bioprinting (extrusion, inkjet, and photolithographic[3]).
PhotoCol® has been used for created 3D microniches[4], as well as for 3D bioprinting human corneas[5].
References:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172302/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243547/
http://www.worldscientific.com/doi/abs/10.1142/S2339547817500091
https://onlinelibrary.wiley.com/doi/abs/10.1002/adbi.201700237
https://www.ncbi.nlm.nih.gov/pubmed/29772228
Product References
References for PhotoCol®:
Isaacson, A., Swioklo, S. & Connon, C. J. 3D bioprinting of a corneal stroma equivalent.Experimental Eye Research173,188–193 (2018).
Kathryn E. Drzewiecki et al, A thermoreversible, photocrosslinkable collagen bio-ink for free-form fabrication of scaffolds for regenerative medicine,TECHNOLOGY(2017).
Drzewiecki, K. E.et al.Methacrylation Induces Rapid, Temperature-Dependent, Reversible Self-Assembly of Type-I Collagen.Langmuir30,11204–11211 (2014).
Gaudet, I. D. & Shreiber, D. I. Characterization of Methacrylated Type-I Collagen as a Dynamic, Photoactive Hydrogel.Biointerphases7,25 (2012).
Izadifar, Mohammad, et al. "UV-assisted 3D bioprinting of nanoreinforced hybrid cardiac patch for myocardial tissue engineering."Tissue Engineering Part C: Methods24.2 (2018): 74-88.
Stoecklin, Celine, et al. "A New Approach to Design Artificial 3D Microniches with Combined Chemical, Topographical, and Rheological Cues."Advanced Biosystems(2018): 1700237.
Mazzocchi, Andrea, et al. "Optimization of collagen type I-hyaluronan hybrid bioink for 3D bioprinted liver microenvironments."Biofabrication(2018).
Nguyen, T. U., Watkins, K. E. & Kishore, V. Photochemically crosslinked cell‐laden methacrylated collagen hydrogels with high cell viability and functionality.Journal of Biomedical Materials Research Part A107,1541–1550 (2019).
Mazzocchi, A.et al.Pleural Effusion Aspirate for Use in 3D Lung Cancer Modeling and Chemotherapy Screening.ACS Biomaterials Science & Engineering5,1937–1943 (2019).
Chhetri, A.et al.Cell Culture and Coculture for Oncological Research in Appropriate Microenvironments.Current Protocols in Chemical Biology11,(2019).
Product Certificate of Analysis
Product Videos
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Video
Telo vs Atelo Collagen
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Neutralization Solution for Easy 3D Collagen Gels
Video
Seeding Collagen Gels with Cells
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4 Key Components for 3D Collagen Gels
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Safety and Documentation
Safety Data Sheet
Certificate of Origin
Declaration of Material Source
Product Disclaimer
This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
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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对于新手来说购买反转录试剂盒是比较理想的，买一个kit看看说明书操作就可以了。推荐两款kit TAKARA和HaiGene，这两款试剂盒都能对RNA样品中的gDNA有效去除，因此对RNA质量的要求不高。TAKARA的RR047A操作方便，反转录温度为37℃，对于大多数试验来讲是满足要求的。HaiGene的D0401操作要多一个步骤，但其反转录温度是55℃（耐高温的反转录酶），提高了反转录温度使得高GC含量、复杂模板、长mRNA的模板都能有效反转录，因此其反转录效率更高，更能够获得样本中基因的真实表达量。如果后续试验是RealTime PCR、ORF克隆、高GC含量、或者你的待研究基因结构复杂程度未知，还是选用耐高温的反转录酶更理想。对于反转录高手来说，直接购买反转录酶、再购买Rnase Inhibitor自己配制反转录体系就可以了。对于样本量大的课题组来讲，相对还是比较经济的。选择反转录酶时仅需要考虑是否需要耐高温的酶，来克服目的基因的复杂结构就可以了。PROMEGA、TAKARA、HaiGene、 TRANSGEN等品牌的反转录酶性价比还都是不错的。Life、NEB的也不错，不过性价比一般，不一一解释了。
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBRGreenMix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBRGreen，并且如果你那是ABI或者Stratagene的PCR如果用SYBRGreen还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊，求推荐！
转录的只是插入的片断，怎么会跟模板一样大呢，一般情况下应该比模板小多了。我都是跑电泳，严格来说应该跑变性的；嫌麻烦可以非变性，快速（防止RNA降解）十分钟即可；抛出来的带会有两条，上面大的是模板，下面一条比较亮的是RNA，那么你的转录就 成功了。
PCR第一个步骤如果没记错的话应该叫“变性”， PCR的话是不需要解旋酶的，直接利用DNA的高温变性特性，在高温下，使氢键断开，DNA双链恢复为单链。 （还有解旋酶好像是解除双螺旋的结构，是一种拓扑异构酶，对于氢键的打开没有贡献吧
加尾法是采用加A酶先对mirna进行加尾，然后再用带oligodt的引物反转录，他的反转录引物是通用的，一次反转录可以获得所有miRNA的cdna，效率高。茎环法是采用特异性反转录引物序列+颈环结构作为反转录引物进行反转录的，一次反转录只能获得一种mirna的cdna，可能有几种一起反转录的，这个我不太清楚一起反转录的效果。然后茎环法的经典即ABI的探针法定量试剂盒。我用过茎环法反转录+染料法定量的，下游引物为通用引物，结果不太好，就现在用的这家的他采用的是双向引物都是特异性的，效果还可以。不知道我说的清楚不，希望能对你有帮助。
如果是用转录出的cDNA做那跟平时就一样了。不需要特别的试剂盒、
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
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