1. Measurements of apparent FRET
Measurements of apparent FRET. (A–C) Representative responses from cell lysates of HEK-293 cells expressing the Epac-based FRET probe (A), CFP (B) or YFP (C).
Measurements of apparent FRET. (A–C) Representative responses from cell lysates of HEK-293 cells expressing the Epac-based FRET probe (A), CFP (B) or YFP (C). Measurements were made as described previously (Leavesley et al., 2013; Rich et al., 2013). In brief, cell lysates were suspended in a stirred cuvette (4 × 106 cells in 4 ml) and excited at 430 nm, then emission intensity was measured from 450 to 650 nm using a spectrofluorimeter. Data were normalized to the peak intensity. (A) The change in apparent FRET response measured at 0 and 10 µM cAMP (black and gray lines, respectively). Note the modest change in FRET associated with a saturating change in cAMP. (B) Changing pH from 7 (blue line) to 6 (black line) had little effect on the measured fluorescence intensity of CFP. (C) Changing pH had a marked effect on the measured fluorescence intensity of YFP. (D and E) Simulations demonstrating the effects of changing pH on apparent FRET measurements and interpretations of the underlying cAMP signals.
Thomas C. Rich et al. J Gen Physiol 2014;143:17-27
© 2014 Rich et al.