Preparation and characterization of the polyclonal antibody against GAR domain of microtubule actin cross-linking factor 1 (MACF1)

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 the polyclonal 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.