Protein Secondary Structure Analysis by CD Spectroscopy
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Corresponding Organization : California State University, Long Beach
Variable analysis
- Reduction of disulfide bonds using 2X molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 24 °C for 16 h
- Ellipticity of freshly re-folded protein samples (0.2 mg/ml protein in 10 mM ammonium bicarbonate, pH 7.4) measured in the far-UV range between 185 nm and 260 nm
- Molar ellipticity ([θ]) and percent α-helix content calculated as described previously [58]
- CD measurements performed on a Jasco 810 spectropolarimeter at 24 °C
- Protein samples measured in a 0.1 cm path length cylindrical cuvette
- Far-UV profiles averaged from four independent scans, recorded at a scan speed of 20 nm/min, with response time of 2 s and bandwidth of 1 nm
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