Takata Takanobu, Murayama Hiroki, Masauji Togen
Department of Life Science, Medical Research Institute, Kanazawa Medical University, Uchinada, Japan.
Department of Pharmacy, Kanazawa Medical University Hospital, Uchinada, Japan.
Bio Protoc. 2024 Jul 20;14(14):e5038. doi: 10.21769/BioProtoc.5038.
Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells. Key features • This protocol leverages the idea that advanced glycation end products are modified proteins. • The lysis buffer containing Tris, urea, thiourea, and CHAPS enables probing proteins onto PVDF membranes. • Intracellular GA-AGE levels may be quantified for some cell types using polyclonal anti-GA-AGE antibodies and standard GA-AGE-modified BSA. • The lysis buffer may be simultaneously prepared with the cell lysate. • There is no limit to the type of cultured cells used in the preparation of cell lysate.
晚期糖基化终末产物（AGEs）是通过糖类（如葡萄糖和果糖）及其中间产物/非酶促产物[如甲基乙二醛和甘油醛（GA）]与蛋白质反应/修饰而形成的。2017年，高田贵信博士等人开发了一种新型狭缝印迹法来定量细胞内源自GA的AGEs（GA-AGEs）。尽管原始方法需要硝酸纤维素膜，但我们推测AGEs中包含的修饰蛋白可能在聚偏二氟乙烯（PVDF）膜上得到有效检测。由于市售裂解缓冲液不适合此目的，高田博士开发了一种狭缝印迹法，使用自制的含有2-氨基-2-羟甲基-1,3-丙二醇（Tris）、尿素、硫脲和3-[(3-胆酰胺丙基)-二甲基铵]-1-丙烷磺酸盐（CHAPS）的裂解缓冲液，该缓冲液能有效地将AGEs检测到PVDF膜上。狭缝印迹法还需要计算Tris、尿素、硫脲和CHAPS的浓度，以及要检测到PVDF膜上的蛋白质和质量。GA-AGE修饰的牛血清白蛋白（BSA，GA-AGEs-BSA）用于绘制标准曲线并对抗GA-AGEs抗体的非特异性结合进行中和，从而能够定量细胞裂解物中的GA-AGEs。本文介绍了C2C12细胞内GA-AGE水平狭缝印迹分析的详细方案。关键特性 • 本方案利用了晚期糖基化终末产物是修饰蛋白这一理念。 • 含有Tris、尿素、硫脲和CHAPS的裂解缓冲液能够将蛋白质检测到PVDF膜上。 • 使用多克隆抗GA-AGE抗体和标准GA-AGE修饰的BSA可对某些细胞类型的细胞内GA-AGE水平进行定量。 • 裂解缓冲液可与细胞裂解物同时制备。 • 制备细胞裂解物所用的培养细胞类型没有限制。
