Cary 300 bio uv vis spectrometer

Cell pellets were resuspended in lysis buffer (10 mM Tris-MgCl₂, EDTA pH 7.0) containing 0.6 mM AEBSF and 8.7 μM lysozme. The treated cells were incubated on ice for 40 min and then sonicated for 30 seconds seven times. The inclusion bodies were collected by centrifugation (7,000 rpm, 30 min, 4 °C). The pellet was washed three times using Tris EDTA (TE) buffer containing 1% triton X-100, and the final pellet was stored at −20 °C. Before purification, inclusion bodies were dissolved in a denaturation buffer (50 mM Tris-HCl, 8 M urea, pH 8.0) and incubated at room temperature for 30 min. The undissolved portions were removed by centrifugation (13,000 rpm, 15 min), and the supernatant was passed through a 0.22 μm filter. An ion exchange column, packed with Q Sepharose Fast Flow strong anion resins (GE Healthcare Life Sciences, Pittsburgh, PA) was used to purify the urea denatured βMPs using a 0 to 0.5 M NaCl gradient in the denaturation buffer [65 (link)]. The elution fractions containing purified protein were determined by SDS-PAGE analysis (Figure S3). OmpF, OmpG and OmpGF were eluted at NaCl concentrations of 75 mM, 200 mM and 160 mM, respectively. The concentrations of purified proteins were determined by absorbance at 280 nm (
${\epsilon }_{280}^{\text{OmpF}}=54,210{\mathrm{M}}^{-1}{\text{cm}}^{-1}$ ,
${\epsilon }_{280}^{\text{OmpG}}=87,780{\mathrm{M}}^{-1}{\text{cm}}^{-1}$ ,
${\epsilon }_{280}^{\text{OmpGF}}=70,250{\mathrm{M}}^{-1}{\text{cm}}^{-1}$ ) using a Cary 300 Bio UV-vis spectrometer (Varian, Inc.).