Department of Molecular Metabolic Regulation Research, Sasaki Institute, Sasaki Foundation, 2-2 Kandasurugadai, Chiyoda-ku, Tokyo, 101-0062, Japan.
Translational Research Center, Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-0022, Japan.
Sci Rep. 2017 Jul 31;7(1):6949. doi: 10.1038/s41598-017-04456-4.
Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.
目前，对于生物体内胰岛素原/胰岛素含量的定量分析往往仅通过酶联免疫吸附测定法（ELISA）进行评估，尽管使用某种定量方法评估结果的充分性很重要。值得注意的是，很少有科学论文使用 Western blot（WB）——另一种免疫测定法——来检测胰岛素原/胰岛素。我们发现通用 WB 进行胰岛素和胰岛素原定量分析时存在两个问题：凝胶电泳中胰岛素带的形状发生扭曲，以及肽激素（主要是胰岛素）对印迹膜的保留效力较低。我们通过优化样品缓冲液中的十二烷基硫酸钠浓度解决了第一个问题，通过用封闭溶液短时间处理后进行戊二醛固定解决了第二个问题。通过对标准品、MIN6c4 细胞裂解物和 MIN6c4 培养上清液中的胰岛素原/胰岛素进行定量，证实了这些改进。此外，我们还表明，经过改良的 WB 适用于其他与糖尿病相关的肽激素：胰岛素类似物、胰高血糖素、GLP-1s、生长抑素、ghrelin 和胰多肽。我们的数据表明，经过改良的 WB 可以通过提供基于电泳的分析信息，为糖尿病相关肽的定性或定量分析做出贡献，尽管 ELISA 是肽激素定量的几乎唯一方法，但它仅提供数值数据。
