Alexa fluor 488 succinimidyl ester

The Fmoc-protected CLE9 analog Fmoc-[Lys²]CLE9 was synthesized by Fmoc chemistry using a peptide synthesizer (Biotage Initiator + Alstra). Alexa Fluor 488 succinimidyl ester (0.3 mg, Thermo Fisher), Fmoc-[Lys²]CLE9 (3.1 mg), and NaHCO₃ (10.0 mg) were dissolved in 250 μl of 50% acetonitrile and stirred for 1 h in the dark at room temperature. Crude peptide was purified by reverse-phase HPLC and lyophilized to yield analytically pure Fmoc-[(Alexa488)Lys²]CLE9. To this purified peptide, 200 μl of 20% piperidine was added in acetonitrile, followed by incubation for 1 h in the dark at room temperature. This deprotected peptide was further purified by reverse-phase HPLC and lyophilized to obtain analytically pure [(Alexa488)Lys²]CLE9 (Alexa488-CLE9). [¹²⁵I]ASA-CLE9 and [¹²⁵I]ASA-[Ara₃]CLV3 were prepared as previously described^{15 (link),17 (link),28 (link)}.

Tubulin was purified from porcine brain by three cycles of depolymerization and polymerization followed by phosphocellulose chromatography^{34 (link)}. Purified tubulin was aliquoted and flash-frozen and stored in liquid nitrogen. For the microbead motility assay, biotinylated (biotin-(AC₅)₂-Sulfo-OSu, Dojindo, Kumamoto, Japan) and Alexa 488-labeled (Alexa Fluor 488 succinimidyl ester, Thermo Fisher Scientific, Waltham, MA) microtubules were prepared by polymerization of a mixture of biotinylated, Alexa488-labeled, and non-fluorescent tubulin in a molar ratio of 15 : 1 : 30–50 in BRB80 buffer (80 mM PIPES, 1 mM MgCl₂, 1 mM EGTA, pH 6.8) supplemented with 1 mM GTP and 1 mM MgCl₂ for 30 min at 37 °C, and then stabilized by adding 40 µM taxol (Sigma-Aldrich, St. Louis, MO)^{28 (link)}. For the in vitro microtubule gliding assay, rhodamine-labeled (X-rhodamine succinimidyl ester, Thermo Fisher Scientific, Waltham, MA) microtubules were prepared by polymerization of a mixture of rhodamine-labeled and non-fluorescent tubulin in a molar ratio of 1 : 16 in BRB80 with 1 mM MgCl₂ and 1 mM GTP for 30 min at 37 °C and then stabilized by adding 20 µM taxol^{35 (link)}.