Fig. 2.

Fluorescence correlation spectroscopy and photon counting histogram analysis of SMALP-purified ABCG2-GFP. SMALPs containing ABCG2-GFP were prepared in assay buffer as described in Methods, and FCS measurements were obtained from 150 μL of sample as described in Methods. (A) Fluorescence fluctuation traces were obtained using 488 nm excitation and analysed by either autocorrelation (AC) analysis or photon counting histogram (PCH) analysis. (B) Autocorrelation curves (upper) were best fit by a 2-component 3D diffusion model (residuals shown in lower) to obtain an average dwell time and particle number of ABCG2-GFP SMALPs, from which a diffusion coefficient and concentration could be calculated. (C) PCH analysis of the same fluctuations (upper) using a 20 μs bin time could be fit (residuals, lower) to obtain a value for the molecular brightness of the ABCG2-SMALPs. Data shown is an example read and analysis of a single sample, representative of those described in Table 2.