Blasius Lynne, Thomas Ezekiel C, Collet Erik H, Jenson Erin, Pearson Chad G, Ohi Ryoma, Verhey Kristen J
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Department of Cell and Developmental Biology, University of Colorado - Anschutz Medical Campus, Aurora, Colorado, USA.
Cytoskeleton (Hoboken). 2025 Jul 12. doi: 10.1002/cm.70011.
Post-translational modifications (PTMs) to tubulin subunits in microtubule filaments are thought to comprise a component of the tubulin code that specifies microtubule functions in cell physiology and animal development. Acetylation of Lysine-40 (K40) on α-tubulin (αTub-K40ac) and glutamylation of both α- and β-tubulin are two tubulin PTMs of interest to the field. Antibodies that recognize these PTMs have been indispensable tools to study the localization of these PTMs as well as their biological functions. Although widely used, these antibodies are procured from commercial sources and thus have drawbacks including availability, high cost, and lack of reproducibility. To mitigate these downsides, we report the protein sequences of GT335 (anti-glutamylation) and 6-11B-1 (anti-αTub-K40ac) monoclonal antibodies and describe the use of these sequences to generate recombinant monoclonal antibody (rMAb) versions of GT335 and 6-11B-1. We demonstrate through western blotting and immunofluorescence of cultured mammalian cells and Tetrahymena thermophila that rMAb-GT335 and rMAb-611B1 match the specific activity of the commercially available antibodies. Our work provides the field with a renewable source of antibodies with high specificity and affinity towards tubulin glutamylation and acetylation and opens the door to more reproducible and large-scale studies of the function and regulation these tubulin PTMs.
微管丝中微管蛋白亚基的翻译后修饰（PTMs）被认为是微管蛋白编码的一个组成部分，该编码在细胞生理学和动物发育中指定微管功能。α-微管蛋白上赖氨酸-40（K40）的乙酰化（αTub-K40ac）以及α-和β-微管蛋白的谷氨酰化是该领域感兴趣的两种微管蛋白PTM。识别这些PTM的抗体一直是研究这些PTM的定位及其生物学功能不可或缺的工具。尽管这些抗体被广泛使用，但它们是从商业来源获得的，因此存在包括可用性、高成本和缺乏可重复性等缺点。为了减轻这些不利因素，我们报告了GT335（抗谷氨酰化）和6-11B-1（抗αTub-K40ac）单克隆抗体的蛋白质序列，并描述了使用这些序列生成GT335和6-11B-1的重组单克隆抗体（rMAb）版本。我们通过对培养的哺乳动物细胞和嗜热四膜虫进行蛋白质印迹和免疫荧光实验证明，rMAb-GT335和rMAb-611B1与市售抗体的比活性相当。我们的工作为该领域提供了一种可再生的抗体来源，该抗体对微管蛋白谷氨酰化和乙酰化具有高特异性和亲和力，并为更具可重复性和大规模研究这些微管蛋白PTM的功能和调控打开了大门。
