Glow discharged carbon coated grid

G-actin (4 µM) was incubated in 0.4 mM ATP, 2 mM Tris, pH 7.5, 0.2 mM CaCl₂, 0.01% NaN₃, and 0.5 mM DTT for 10 min on ice. Polymerization buffer (20X) was added to G-actin to reach a final concentration of 1X (100 mM KCl, 25 mM imidazole, pH 8.0, 2 mM MgCl₂, 1 mM EGTA, and 0.1 mM ATP), and actin was polymerized for 30 min at room temperature. F-actin was kept at 4°C for several hours during TEM imaging. From the same batch of prepared F-actin, all TEM samples (including the actin-only control) were prepared. Wild-type Tpm1.1 (1.25 µM) or 2 µM Tpm1.1[R21H] was added to F-actin and the mixture was incubated at 4°C for 10 min. Lmod2 (0.9 µM) was added to F-actin in the absence or presence of Tpm1.1 and incubated at room temperature for 10 min.
To visualize complexes of F-actin with Lmod2 and with or without bound Tpm1.1, aliquots (7 µl) were applied to glow-discharged carbon-coated grids (Electron Microscopy Sciences, Hatfield, PA) and stained with 2% uranyl acetate. The grids were examined in a JEOL 1200 EXII transmission electron microscope (JEOL USA, Peabody, MA) under regular dose conditions at an accelerating voltage of 70 keV.
Pellet samples from cosedimentation assays were run on 10% polyacrylamide SDS gels. The gels were scanned using ChemiDoc XRS (Bio-Rad, Hercules, CA) and quantified using the National Institutes of Health ImageJ software.

Skeletal striated muscle G-actin (2.5 µM) was polymerized in 10 mM Hepes buffer, pH 7.4, 100 mM KCl, 1 mM MgCl₂, 0.5 mM DTT, and 0.5 mM ATP for 2 h. Actin filaments were incubated with 14 µM His-SEPT6 or His-SEPT9 for 15–30 min at 4°C. To visualize complexes of F-actin with septins, aliquots (10 µl) were applied to glow-discharged carbon-coated grids (Electron Microscopy Sciences) and stained with 2% uranyl acetate. The grids were examined in a transmission electron microscope (TEM; 1200EXII; JEOL Inc.) under regular dose conditions at an accelerating voltage of 70 keV. The images shown in Fig. 3 (F and G) are representative of similar results obtained from three independent experiments.