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An experimental toolbox for characterization of mammalian collagen type I in biological specimens.
Nature Protocols ( IF 13.1 ) Pub Date : 2018-Mar-01 , DOI: 10.1038/nprot.2017.117
Héctor Capella-Monsonís , João Q Coentro , Valeria Graceffa , Zhuning Wu , Dimitrios I Zeugolis

Collagen type I is the most abundant extracellular matrix protein, and collagen type I supramolecular assemblies (e.g., tissue grafts, biomaterials and cell-assembled systems) are used extensively in tissue engineering and regenerative medicine. Many studies, for convenience or economic reasons, do not accurately determine collagen type I purity, concentration, solubility and extent of cross-linking in biological specimens, frequently resulting in erroneous conclusions. In this protocol, we describe solubility; normal, reduced and delayed (interrupted) SDS-PAGE; hydroxyproline; Sircol collagen and Pierce BCA protein; denaturation temperature; ninhydrin/trinitrobenzene sulfonic acid; and collagenase assays and assess them in a diverse range of biological samples (e.g., tissue samples; purified solutions or lyophilized materials; 3D scaffolds, such as sponges and hydrogels; and cell media and layers). Collectively, the described protocols provide a comprehensive, yet fast and readily implemented, toolbox for collagen type I characterization in any biological specimen.

中文翻译:

实验工具箱,用于表征生物样本中的I型哺乳动物胶原蛋白。

I型胶原蛋白是最丰富的细胞外基质蛋白,I型胶原蛋白的超分子组装体(例如组织移植物,生物材料和细胞组装系统)广泛用于组织工程和再生医学中。出于方便或经济原因,许多研究未能准确确定生物样品中I型胶原蛋白的纯度,浓度,溶解度和交联程度,经常会得出错误的结论。在此协议中,我们描述了溶解度;正常,减少和延迟(中断)的SDS-PAGE;羟脯氨酸 Sircol胶原蛋白和Pierce BCA蛋白;变性温度 茚三酮/三硝基苯磺酸; 和胶原酶分析,并在各种生物样品(例如组织样品;纯化的溶液或冻干的材料; 3D支架,如海绵和水凝胶;以及细胞培养基和层)。总体而言,所描述的方案为任何生物学标本中的I型胶原蛋白表征提供了一个全面而又快速且易于实施的工具箱。
更新日期:2018-02-23
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