X band cw epr spectrometer

CW EPR spectra of EHD2 in solution at a concentration of approximately 2 g/L were recorded on a Bruker X-band CW EPR spectrometer fitted with an ER4119HS resonator at 12.7 mW incident microwave power at room temperature. Liposome-bound samples were produced by incubating 50 μg of spin labeled protein with 1.5 mg Folch-SUV (1:30 w/w protein to lipid ratio) at room temperature (RT) for 20 min. SUV-bound EHD2 was separated from unbound EHD2 by centrifugation (152,800 g, 20 min, 22 °C). The pellet was resuspended in 20 mM HEPES, pH 7.5, 300 mM NaCl and spectra were recorded as described above. Accessibilities to O₂ (from air, Π_Ox) and 10 mM NiEDDA (Π_NiEDDA) were obtained from power saturation experiments using a Bruker ER4123D dielectric resonator. The depth parameter Φ was calculated from Φ = ln[Π_Ox/Π_NiEDDA] (Altenbach et al., 1994 (link)). The membrane insertion depth was obtained by calibrating Φ using N-tempoylpalmitamide (gift from Kyoung Joon Oh) for position 0 and 1-palmitoyl-2-stearoyl-(n-DOXYL)-sn-glycero-3-phosphocholine (Avanti Polar Lipids) with spin labels attached at carbon 5, 7, 10 and 12 (Altenbach et al., 1994 (link)).