![Figure 1. Time needed to attain equilibrium for AlexaFP binding to D71A located on the bottom surface of live CHO cells. (A) Schematic drawing of D71A bound by AlexaFP. n, norleucine. (B) A representative time series of TIRF images, observed at 0, 20, 60, and 180 s after applying 1 nM AlexFP. Orange lines indicate the perimeters of the two cells found in this view field. The punctate appearance is probably caused by the presence of dimers, incidentally overlapped monomers within the spatial resolution limit, and statistical variations. (C) Time-dependent increases in the fluorescence intensities of the surface-bound AlexaFP in the area of 10 × 10 µm, after applying 1 (top) and 6 nM (bottom) AlexaFP. The y axis is normalized using the saturation value for each concentration, but the absolute saturation value for the 1-nM experiment is ∼0.43× of that for the 6-nM experiment. Red curves show the best fit functionI(t) = CN {1 − exp[−k[c]t]} ([c], AlexaFP concentration; fitting parameters, CN = ∼1; k, binding rate constant), with a k of 0.0076 ± 0.0017 s−1nM−1 (five independent determinations), yielding the exponential time constant for 6 nM AlexaFP of 22 ± 4.1 s. Note that each value determined in this paper is given as the mean ± standard error (error bars), and, in the case of the fitting parameters, the fitting error at the 68.3% confidence limit is given. This result clearly indicates that the ligand quickly enters the space between the bottom membrane and the coverslip, and that the AlexaFP binding at 6 nM had already achieved the equilibrium conditions at 6–8 min after its application (all of the observations were performed during this period). Furthermore, the ligand binding in the central region of the bottom membrane occurs as fast as that in the peripheral region (Fig. 1 B), which suggests that the ligand concentration within the space between the bottom membrane and the coverslip is rapidly equalized with that in the bulk space (indeed, one of the major reasons we selected CHO cells for this study is this fast entrance of the ligand in the space between the bottom membrane and the coverslip. For some cell types, the ligand reaches the central part of the bottom membrane quite slowly).](https://cdn.rupress.org/rup/content_public/journal/jcb/192/3/10.1083_jcb.201009128/5/jcb_201009128r_rgb_fig1.jpeg?Expires=1771763895&Signature=BCv3pf3xBuHZ2zfCpCFwYI0ZYRiV8LokDQpc69DZizpJddumn4~SStKIDbsNVtK9~7BY~IKkTssdAgQPxEnEDBia89FJzcoBwHEbEs913nKjJhQnSvrBPpwucqkwpUogkxIjIB~HctihrUnkb4DZS9XstIYwBqqgzZ2lXLOUfQ3vE5Hpzcr3MLkzULOEpvB6~d0OhJUEAM~F2IgGZzuvAHpe8jPsW-1o-gqnAW2FlXBJDCeWC4Qd2IrltTMnn3on3DrMqjwDxspNZM7TbQg02V8dvU9vOl2DLZo4VCiEqkQEYAq2uALpyzG9axhQT4arcZ3nYKU4a11y0VkGKsKC2w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Time needed to attain equilibrium for AlexaFP binding to D71A located on the bottom surface of live CHO cells. (A) Schematic drawing of D71A bound by AlexaFP. n, norleucine. (B) A representative time series of TIRF images, observed at 0, 20, 60, and 180 s after applying 1 nM AlexFP. Orange lines indicate the perimeters of the two cells found in this view field. The punctate appearance is probably caused by the presence of dimers, incidentally overlapped monomers within the spatial resolution limit, and statistical variations. (C) Time-dependent increases in the fluorescence intensities of the surface-bound AlexaFP in the area of 10 × 10 µm, after applying 1 (top) and 6 nM (bottom) AlexaFP. The y axis is normalized using the saturation value for each concentration, but the absolute saturation value for the 1-nM experiment is ∼0.43× of that for the 6-nM experiment. Red curves show the best fit functionI(t) = CN {1 − exp[−k[c]t]} ([c], AlexaFP concentration; fitting parameters, CN = ∼1; k, binding rate constant), with a k of 0.0076 ± 0.0017 s−1nM−1 (five independent determinations), yielding the exponential time constant for 6 nM AlexaFP of 22 ± 4.1 s. Note that each value determined in this paper is given as the mean ± standard error (error bars), and, in the case of the fitting parameters, the fitting error at the 68.3% confidence limit is given. This result clearly indicates that the ligand quickly enters the space between the bottom membrane and the coverslip, and that the AlexaFP binding at 6 nM had already achieved the equilibrium conditions at 6–8 min after its application (all of the observations were performed during this period). Furthermore, the ligand binding in the central region of the bottom membrane occurs as fast as that in the peripheral region (Fig. 1 B), which suggests that the ligand concentration within the space between the bottom membrane and the coverslip is rapidly equalized with that in the bulk space (indeed, one of the major reasons we selected CHO cells for this study is this fast entrance of the ligand in the space between the bottom membrane and the coverslip. For some cell types, the ligand reaches the central part of the bottom membrane quite slowly).
