Figure 3
From: NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes
Protein concentrations and micellar concentrations of extraction and destination detergents during detergent exchange on the Ni-NTA column. Incomplete (A) and successful (B) exchange of FC14 with DDM. Detergent micellar concentrations are shown in green (FC14) and orange (DDM); protein concentrations are shown in blue; the applied micellar concentration of DDM is shown in magenta. (C) 1H-NMR spectra (5.5...4.9 ppm and 3.4...-0.5 ppm regions) of the wash and elution fractions in B, collected during the exchange of FC14 with DDM on a Ni-NTA column: cyan – 1 column volume (cv) wash; magenta – 5 cv wash; blue – 10 cv wash; green – 12 cv wash; red – 16 cv wash and black – 1 cv elution. The spectra are normalized according to the integral intensity of the DSS (CH3)3-Si-group signal (0.0 ppm) and stacked for comparison. The FC14 concentration was calculated based on the integral of the (CH3)3-N- signal at 3.23 ppm. The DDM concentration was calculated based on the integral of the C1H signal at 5.34 ppm for washes 3, 5 and 6 and the integral of -(CH2)n-CH3 peak for wash 8 and elution 1, to which contribution from FC14 was known based on the integral of the (CH3)3-N-signal.