In vitro microtubule-binding assays

1、Remove the microtubule protein from the -80℃ refrigerator and dissolve on ice. 

2、Mix the appropriate amount of microtubule protein with the BRB80 solution (100 mM PIPES, 1 mM MgSO4, 2 mM EGTA, 1 mM GTP, pH 6.8) and place on ice for 5 minutes.

3、Then transfer the reaction system to a metal bath at 37℃ and incubate for 30 minutes.

4、Stop the reaction with BRB80 buffer (20 uM).

5、The polymerized microtubules were pelleted  by centrifugation at 45 000 rpm for 45 min at 37 °C.

6、Remove the supernatant and resuspend the precipitation (polymerized microtubules) with  BRB80 buffer (20 uM).

7、GST-labeled proteins are expressed in the E. coli BL21 strain for 16 hours.

8、E. coli collection is re-suspended and lysed with GST lysis buffer (0.5% Tween-20, 20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EGTA, pH 7.5)。

9、Enrich the GST label protein with Glutathione Sepharose 4B beads.

9、Wash the beads with GST lysis buffer 3 times and then wash the beads with BRB80 buffer  3 times.

10、Incubated the GST beads with polymerized microtubules for 30 minutes at 37 °C。

11、Wash the beads 5 times with BRB80 buffer and then added 60 ul 1 × loading buffer, boil the sample at 100°C for 8 minutes to prepare the sample.

12、The sample is analyzed by SDS-PAGE followed by Western Blot.