Otespa

tsA 201 cells expressing appropriate combinations of Orai1-Myc-His, σ1R-FLAG, and HA-STIM1 were treated with thapsigargin, followed by biotin-sulfo-NHS, and then purified using sequential avidin and anti-Myc affinity chromatography, as described in the Isolation of surface biotinylated proteins section. About 45 µl of proteins was added to a 1-cm² mica disk, incubated at 20°C for 10 min, gently washed with water, and dried under nitrogen. Samples were imaged in air using an atomic force microscope (Multimode; Bruker). The silicon cantilever (OTESPA; Bruker) was set at a drive frequency of 271–321 kHz and spring constant of 12–103 N/m. The scan rate was 3 Hz, and the applied imaging force was kept as low as possible (target amplitude of 1.0 V and amplitude set point of 0.7–1.0 V). Molecular volumes for individual particles were determined using an image processor (version 5; Scanning Probe). For particles within complexes, particle heights (h) and radii (r) were measured manually using Nanoscope software. Particle volumes (V_m) were then calculated from ${\mathrm{V}}_{\mathrm{m}}=\mathrm{ }\frac{\mathrm{\pi h}(3{\mathrm{r}}^2+{\mathrm{h}}^2)}{6}\mathrm{.}$ Molecular volume (V_c), based on a known molecular mass (M₀), was calculated from ${\mathrm{V}}_{\mathrm{c}}\mathrm{ }=\mathrm{ }\frac{{\mathrm{M}}_0({\mathrm{V}}_1+{\mathrm{dV}}_2)}{{\mathrm{N}}_0}\mathrm{,}$ where N₀ is Avogadro’s number, V₁ is the specific particle volume (0.74 cm³/g), V₂ is the water specific volume (1 cm³/g), and d is the extent of hydration (assumed to be 0.4 g H₂O/g protein).