FIGURE 2

Spectroscopic and biochemical characterization of phiLOV3, TagRFP658, and miRFP2 in comparison to their precursors. (a?c) Absorption and fluorescence emission spectra of (a) phiLOV2.1 (dashed line) and phiLOV3 (solid line), (b) TagRFP657 (dashed line) and TagRFP658 (solid line), and (c) miRFP (dashed line) and miRFP2 (solid line). (d) Photobleaching curves of phiLOV2.1 (dashed line) and phiLOV3 (solid line) expressed in HEK293FT cells (n = 18 cells from 1 transfected sample, each). Photobleaching conditions: excitation 475/34 nm from an LED at ~5 mW/mm². (e) Photobleaching curves of TagRFP657 (dashed line) and TagRFP658 (solid line) expressed in HEK293FT cells (n = 16 cells from 1 transfected sample, each). Photobleaching conditions: excitation 628/31BP from an LED at 95 mW/mm². (f) Photobleaching curves of miRFP (dashed line) and miRFP2 (solid line) expressed in primary cultured mouse neurons (n = 25 and 61 cells, respectively, from one culture, each). Photobleaching conditions: excitation 628/31BP from an LED at 88 mW/mm². (g?i) pH dependence of fluorescence for (g) phiLOV2.1 (dashed line) and phiLOV3 (solid line), (h) TagRFP657 (dashed line; data from Reference 32 ) and TagRFP658 (solid line), and (i) miRFP (dashed line; data from Reference 35) and miRFP2 (solid line). (j?l) Size-exclusion chromatography of (j) phiLOV3 at a concentration of 3.8 mg/ml, (k) TagRFP658 at a concentration of 8 mg/ml, and (l) miRFP2 at a concentration of 8.8 mg/ml (solid lines) and the indicated molecular weight standards (dashed lines)