Mwco pierce protein concentrators

The overall secondary structure of purified EgtU SBD was analyzed by CD using an Applied Photophysics Chirascan V100 instrument. Purified protein was buffer-exchanged from HEPES into PBS using 3 kDa MWCO Pierce^™ Protein Concentrators (Thermo Fisher) via centrifugation for 30-min intervals at 4 °C and 15,000 × g. Protein was either used directly, or if thawed from frozen, centrifuged at 4 °C for 20 min at 20,000 × g to pellet any precipitate. A sample of supernatant was taken and denatured in 7.5 M guanidine hydrochloride and absorbance at 280 nm was measured. The concentration was determined using Beer’s law with an extinction coefficient of 27,390 M⁻¹ cm⁻¹. The protein was diluted to 15 μM in 400 μL total volume of PBS and loaded into a 1-mm path length quartz cuvette (Starna Cells). CD spectroscopy absorbance measurements were conducted at 20 °C from 190 – 300 nm wavelengths with a 1-nm step and 2-sec averaging time, resulting in 80,000 independent 25-μsec measurements. Measurements were performed in duplicate for each mutant, and data were corrected using a PBS blank. The raw absorbance (millidegrees, θ) was converted to Mean Residue Ellipticity ([θ]MR) using the following equation: [θ]MR = (100 × θ) / ((C × N) × I), where C is the molar protein concentration, N is the number of amino acids, and I is the cell path length in centimeters (Greenfield, 2006 (link)).