Abstract

Growth arrest-specific 2 protein (Gas2) related domain (GAR domain), located at the C-terminal of microtubule actin cross-linking factor 1 (MACF1), plays a key role in microtubules (MTs) binding. To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR domain. Polyclonal antibody was then developed by inoculation of New Zealand rabbit with the recombinant GAR protein (rGAR) (97% purity). After several doses of immunization, the titer of antiserum reached 1: 62500 when evaluated by ELISA. The polyclonal antibody was further confirmed by Western blot, which gave results of high specificity and sensitivity (1: 5000). By using thepolyclonal antibody to detect MACF1’s association with MTs, laser scanning confocal microscopy (LSCM) showed that widely expressed MACF1 was partially aligned along MT filaments in the cytoplasm and co-located with MTs. This polyclonal antibody will be a valuable tool for the further studies of MACF1 and its GAR domain.

Key words: Microtubule actin cross-linking factor 1 (MACF1), GAR domain, clone, expression, purification, polyclonal antibody.