Surface-limited reactions for spatial control of kinesin–microtubule motility assays using indirect irradiation of an electron beam

B. Solution preparation for an MT motility assay

Buffer solutions for coating kinesins on an SiN membrane were prepared based on the literature.2828. K. Hatazawa, H. Miyazako, R. Kawamura, and T. Hoshino, Biochem. Biophys. Res. Commun. 514, 821 (2019). https://doi.org/10.1016/j.bbrc.2019.04.200 The preparation protocol was summarized as follows. First, a 2 × BRB buffer was prepared with concentrations of 160 mM PIPES, 2 mM EGTA, and 2 mM MgCl2 whose pH was adjusted at 6.8 by adding a KOH aqueous solution. A premix buffer (premix) was prepared with 1 μl of 100 mM GTP, 2 μl of 200 mM MgCl2, 10 μl of 2 × BRB, 5 μl of DMSO, and 2 μl of Milli-Q water. For coating anti-GFP antibodies, antibody buffer (AB) was prepared with 2 μl of a 2 mg/ml anti-GFP antibody solution, 10 μl of 2 × BRB, and 8 μl of Milli-Q water. For coating casein, wash buffer (WB) was prepared with 25 μl of an approximately 10 mg/ml casein solution, 50 μl of 2 × BRB, and 25 μl of Milli-Q water. For coating kinesins, a K560-GFP solution (KG) was prepared with 5 μl of a 180 nM K560-GFP solution, 1 μl of approximately 10 mg/ml casein solution, 10 μl of 2 × BRB, 2.5 μl of 10 mM TCEP, and 6.5 μl of Milli-Q water. Mot-mix buffer was prepared with 500 μl of 2 × BRB, 245 μl of Milli-Q water, 50 μl of casein solution, 5 μl of 200 mM MgCl2, 100 μl of 10 mM TCEP, 10 μl of 20 000 U/ml catalase solution, 10 μl of 1000 U/ml glucose oxidase solution, and 10 μl of 2.5 M D-glucose in Milli-Q water. For rinsing the membrane, M_BRB buffer was prepared with 186 μl of Mot-mix, 12 μ of Milli-Q water, and 2 μl of 1 mM paclitaxel. For the movement of kinesins, M buffer was prepared with 558 μl of Mot-mix, 6 μl of Milli-Q water, 6 μl of 1 mM paclitaxel, and 30 μl of 100 mM ATP in Milli-Q water adjusted at pH 7.0 by adding an NaOH aqueous solution.

A488-tubulin solution (AT) was prepared with 4 μl of 30 μM tubulin mixture (tubulin: A488-tubulin = 10:1) and 1 μl of premix. To polymerize MTs, 5 μl of AT was incubated in a water bath at 37 °C for 30 min and then suspended in 200 μl of BRB80P, which was prepared by mixing 100 μl of 2 × BRB, 2 μl of 1 mM paclitaxel in DMSO, and 98 μl of Milli-Q water. AMT was prepared by diluting the suspended AT two to four times with Mot-mix and paclitaxel.

留言 (0)

沒有登入
gif