Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Molecules. 2022 Jul 29;27(15):4867. doi: 10.3390/molecules27154867.
Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).
解读蛋白质翻译后修饰 (PTM) 密码是我们这个时代最大的生化挑战之一。磷酸化和泛素化是决定蛋白质功能、识别、亚细胞定位、稳定性、周转率和命运的关键 PTM。因此，它们的调节失败会导致各种疾病。化学蛋白质合成允许制备泛素化和磷酸化蛋白质，以详细研究它们的生化特性。然而，在完整细胞或细胞提取物中监测这些修饰主要依赖于抗体，而抗体通常具有非靶向结合。在这里，我们报告说，最广泛用于检测丝氨酸 65 磷酸化泛素 (pUb) 的抗体 (Ub) 具有在有丝分裂过程中出现的显著非靶向。这些非靶标与 polo 样激酶 1 (PLK1) 介导的细胞周期相关蛋白和后期促进复合物亚基 1 (APC1) 的磷酸化有关。
