5.5. Binding assay with AMP-PNP or ATP

MTs polymerized as described above (100 µg protein) were incubated with microsomes (40 µg protein) for 30 min at room temperature (+MT). The microsomes had previously been incubated for 15 min with buffer, 5 mM AMP-PNP or 1 mM ATP and 5 µM taxol. Stripped microsomes (see above) were incubated with AMP-PNP. +/−MT-microsome samples were then centrifuged for 1 h at 33 300 r.p.m. in a Beckman SW60 rotor, at 20°C through a 1.2 M sucrose cushion to separate MTs bound to organelles from those not bound to organelles. Three different fractions were collected: soluble fraction (S), organelles lying on the cushion (I) and pelleted MTs and organelles (P). In parallel, a control sample without MTs (−MT) was treated as described for MT/microsome incubation. The P fractions with and without MTs (P +MT and P –MT, respectively) were used for TEM analysis or denatured for electrophoresis. Binding experiments carried out using AMP-PNP were repeated 25 times. Seven and 10 independent experiments were performed by using ATP and stripped microsomes, respectively. About 100 TEM images were taken for each experiment.