Caγ-TuSC has lower propensity to self-oligomerize than the S. cerevisiae γ-TuSC. (A, left) CBB-stained gel of purified Scγ-TuSC and Caγ-TuSC. (right) Molecular ratio of CaTub4 and CaSpc97-CaSpc98 based on intensities. Error bars are SEM. n = 5 independent experiments. (B) Negative-stained Caγ-TuSC and Scγ-TuSC have similar structures under EM single-particle analysis. Boxed negative-stained particles were classified and averaged. See Fig. S2 B for representative micrographs. Bars, 10 nm. (C) Scγ-TuSC has higher oligomerization propensity than Caγ-TuSC. Scγ-TuSC and Caγ-TuSC were analyzed by gel filtration (Superose 6 PC3.2/30). Fractions were analyzed with SDS-PAGE and CBB. (D) As in Fig. 2 C but in the low-salt BRB80 buffer, pH 6.8. (E) Caγ-TuSC was incubated with BRB80 buffer, from 100 to 200 mM KCl, and then subjected to gel filtration. (F) Self-oligomerized Caγ-TuSC in low-salt BRB80 buffer (100 mM KCl) showed ordered spherical structures of 30 nm in diameter. Protein peak (0.9 ml) of E was analyzed by negative-staining EM. Bar, 50 nm. (G) Scγ-TuSC formed high oligomeric assemblies in BRB80 (150 mM KCl) but formed less high-mol-wt oligomers in BRB80 (200 mM KCl). SDS-PAGE with CBB. (H) Summary of buffer effect on γ-TuSC oligomerization. mAU, milli-absorbance unit.