three times microtubular protein was prepared from goat brains by two cycles of temperature dependent assembly disassembly process in pem buffer 100 mm pipes ph 6 9 1 mm mgcl 2 amp 1 mm egta with 1 mm gtp at 37 u00b0 c for fee polymerization experiments it was followed by one more cycle in pem tubulin was purified from 2x mtp using glutamate buffer for assembly mtp polymerization in fee presence or absence of dat1 was measured by the time course of fee turbidity at 37 u00b0 at 345 nm a shimadzu uv 1601 double beam spectrophotometer fitted with a temperature controlled circulating water bath was used for this purpose hela cells were incubated with the drug for 24 h at 37 u00b0 c washed with pbs and fixed with 4 paraformaldehyde at 4 u00b0 c subsequently they were washed and permeabilized with 0 2 triton x 100 in pbs for 20 min at 37 u00b0 c microtubules were stained by a mouse monoclonal antibody against u03b2 tubulin in 1 100 dilution followed by a rhodamine conjugated goat antimouse antibody in 1 50 dilution and were observed by a nikon eclipse te300 microscope emission spectra of dat1 in presence of tubulin were recorded from 400 nm to 600 nm using an excitation wavelength of 374 nm excitation and emission bandpasses are 2 5 nm each and the fluorescence values recorded are uncorrected all fluorescence measurements were performed in a perkin elmer model ls50b luminescence spectrometer the binding parameters of dat1 binding to tubulin.