Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Calle del Arzobispo Morcillo 4, 28029, Madrid, Spain.
Anal Bioanal Chem. 2013 Aug;405(21):6863-71. doi: 10.1007/s00216-013-7101-0. Epub 2013 Jun 19.
The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).
组织中胶原蛋白含量的评估在生物医学研究中很重要，因为这种蛋白质在许多疾病中都会发生改变。羟脯氨酸和 Sirius red 测定法是胶原蛋白定量最常用的方法。然而，这些程序存在一些缺陷，例如需要无氧介质或昂贵的设备、大样本量，或者分别不适合水解胶原蛋白。我们的目标是开发一种特定的、通用的、易于使用的定量方法，适用于从小组织样本和弹性蛋白纯化中获得的提取物，因此适用于同时定量弹性蛋白。该方法基于 Sirius red 与固定在 PVDF 膜上的样品中存在的胶原蛋白的结合，如斑点印迹技术，并通过扫描仪和图像分析软件进行定量。优化了样品加载、Sirius red 浓度、温度和孵育时间、标准物质类型、白蛋白干扰和定量时间。该方法能够定量（1）几种大鼠组织匀浆中的完整胶原蛋白，包括小阻力动脉，（2）来自 NaOH 提取物的部分水解胶原蛋白，与弹性蛋白纯化兼容，以及（3）用于检测高血压和正常血压大鼠之间胶原蛋白含量的差异。我们得出结论，由于该技术具有通用性（定量完整和水解胶原蛋白）、需要小样本量、易于使用（成本低、使用方便、毒性材料少、测试时间短）和特异性（与血清白蛋白的干扰最小），因此可以广泛应用。
