Method for the estimation of the amount of acetylated tubulin

CARBAJAL A, CHESTA ME, BISIG, CG, ARCE CA.

Mendoza

Congreso; Sociedad de Investigaciones en Bioquímica y Biología Molecular; 2012

SAIB

Tubulin, the main protein constituent of microtubules, is subjected to acetylation/deacetylation at the epsilon-amino group of Lys40 of the alpha-chain. Studies on the physiological role of this postranslational modification by many laboratories produced conflictive results, in part due to the unavailability of a method to quantify the amount of acetylated tubulin (AcTubulin) in the corresponding experimental systems. We present a simple method to estimate the percentage of AcTubulin with respect to total tubulin. The method is based on the comparison of the amount of acetylated tubulin (as determined by Western blot or ELISA using a commercial antibody specific to acetylated tubulin) before and after chemical acetylation with acetic anhydride. The amount of AcTubulin is calculated as the ratio of AcTubulin before acetylation over AcTubulin after acetylation, and multiplied by 100. Validation of the method was done by applying it on preparations of known AcTubulin concentrations