Pokidysheva Elena N, Sales Jennifer Diaz, Yagi Shinomi, Ueno Tomonori, Sasai Kanako, Makarenko Alice, Bächinger Hans Peter, Mizuno Kazunori, Boudko Sergei P
Nephrology Division, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Aspirnaut Program, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Matrix Biol Plus. 2025 May 15;26:100174. doi: 10.1016/j.mbplus.2025.100174. eCollection 2025 Jun.
Collagens are a diverse family of proteins present in the extracellular matrix (ECM) of all animals. They play crucial roles in providing structural support to tissues, forming scaffolds for ECM suprastructures, and signaling cells. Certain collagen-binding proteins from pathogenic bacteria, such as CollageN Adhesin (CNA) from , interact with the collagen triple helix to promote host invasion. The extracellular portion of CNA, known as CNA35, which has a molecular weight of 35 kDa, has been used for , , and staining of various collagen types in tissues. Detecting various types of collagens necessitates the use of type- and specie-specific antibodies, which typically exhibit weak affinities for the triple helical regions of collagens. Additionally, the fragmentation of collagens can lead to a loss of detection due to the limited number of available epitopes. Furthermore, antibodies can be expensive, require secondary identification methods, and are often suitable for either immunohistochemistry or western blotting. Although successful procedures for staining collagens in tissues have been implemented, the detection of collagens and their fragments using CNA35 has not been reported for protein blots. In this study, we examined the detection capabilities of a trimeric form of CNA35 for protein blots following SDS-PAGE. We successfully tested collagens I through VI, as well as fragments of collagen IV, under various conditions. Additionally, we investigated the impact of blocking solutions, incubation time, ligand concentration, and CNA35 concentration on sensitivity. We achieved superior detection of all tested collagens and collagen IV fragments, including the 7S domain, which is a highly crosslinked complex composed of four triple-helical strands. The method we developed serves as a universal tool for detecting collagens and collagen-containing peptides in protein blots. It offers several advantages, including sub-nanogram sensitivity, low cost, and compatibility with standard western blotting techniques.
胶原蛋白是存在于所有动物细胞外基质（ECM）中的一类多样的蛋白质家族。它们在为组织提供结构支持、形成ECM超结构支架以及信号传导细胞方面发挥着关键作用。来自致病细菌的某些胶原蛋白结合蛋白，如来自[具体细菌名称未给出]的胶原蛋白黏附素（CNA），与胶原蛋白三螺旋相互作用以促进宿主入侵。CNA的细胞外部分，称为CNA35，分子量为35 kDa，已用于组织中各种胶原蛋白类型的[具体用途未明确给出]、[具体用途未明确给出]和染色。检测各种类型的胶原蛋白需要使用类型和物种特异性抗体，这些抗体通常对胶原蛋白的三螺旋区域亲和力较弱。此外，胶原蛋白的片段化可能由于可用表位数量有限而导致检测失败。此外，抗体可能很昂贵，需要二级鉴定方法，并且通常仅适用于免疫组织化学或蛋白质印迹法。尽管已经实施了在组织中染色胶原蛋白的成功程序，但尚未有关于使用CNA35检测蛋白质印迹中的胶原蛋白及其片段的报道。在本研究中，我们检查了三聚体形式的CNA35在SDS-PAGE后对蛋白质印迹的检测能力。我们在各种条件下成功测试了I型至VI型胶原蛋白以及IV型胶原蛋白的片段。此外，我们研究了封闭液、孵育时间、配体浓度和CNA35浓度对灵敏度的影响。我们对所有测试的胶原蛋白和IV型胶原蛋白片段，包括由四条三螺旋链组成的高度交联复合物7S结构域，都实现了卓越的检测。我们开发的方法是一种用于检测蛋白质印迹中胶原蛋白和含胶原蛋白肽的通用工具。它具有几个优点，包括亚纳克灵敏度、低成本以及与标准蛋白质印迹技术的兼容性。
