Recombinant Monoclonal Antibodies for Detecting the Tubulin Post-Translational Modifications Glutamylation and Lysine-40 Acetylation.

Abstract

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.