Production and Characterization of a Monoclonal Antibody Against P60-Katanin

Abstract

Katanin is a microtubule severing protein composed of two subunits, P60 and P80. The P60 subunit severs microtubules and P80 controls the microtubule severing activity of P60. Katanin promotes spindle shortening and severing processes in mitotic and meiotic spindles. In neurons, the level of P60-katanin was found to be very high in actively growing axons and at the tips of growing neuronal processes and in dendritogenesis. Inhibition of P60-katanin increases microtubule length throughout the neuronal cell body and mitotic/meiotic spindles, indicating that katanin is one of the key proteins for organizing microtubular structure in a variety of cell types. In this study, we produced 1G6, the first monoclonal antibody against P60-katanin. Recombinant P60-katanin (RecP60) protein, which was produced based on a specific region of rat P60-katanin, was expressed in Escherichia coli and used as antigen. 1G6 recognized the endogenous P60-katanin in Western blot analysis and immunocytochemistry in which monoclonal antibody would increase the range of applications. Since P60-katanin polyclonal antibodies have been used in studies until now, obtaining the hybridoma cell, the unlimited source of a monoclonal antibody against P60, results in an advantage in investigating P60-katanin functions.