Table 1.
In vitro properties of the GAF-CaMP2 calcium indicator compared to permanently fluorescent smURFP and GAF-FP proteins.
| Properties | Proteins | |||||||
|---|---|---|---|---|---|---|---|---|
| GAF-CaMP2 | GAF-CaMP2-sfGFP | smURFPa | GAF-FPb | |||||
| apo | sat | apo | sat | |||||
| Absorbance maximum (nm) | 642 (384) | 650 (384) | 630 (383, 490) | 649 (383, 490) | 642 * | 637 (379) | ||
| Excitation maximum (nm) | 620 (371) | 640 (374) | 630 (372, 493) | 642 (374, 493) | 642 | 635 | ||
| Emission maximum (nm) | 676 | 674 | 676 (514) | 674 (514) | 670 | 670 | ||
| Quantum yield (%) c | 1.8 ± 0.2 | 4.8 ± 0.2 | 3.3 ± 0.1 | 6.9 ± 0.5 | 17.9 ± 0.2 * | 7.3 | ||
| ε (mM−1·cm−1) d | 22.2 ± 0.2 | 29.2 ± 0.3 | 15.7 ± 0.4 | 27.5 ± 0.9 | 180 | 49.8 | ||
| Brightness vs. EGFP (%) e | 1.2 | 4.2 | 1.5 | 5.6 | 96 | 11 | ||
| pKa | 5.16 ± 0.05; 6.99 ± 0.06; ≥9.3 ± 0.3 | 4.89 ± 0.05; 7.15 ± 0.02; ≥9.1 ± 0.2 | 5.30 ± 0.02; 7.28 ± 0.02; ≥9.28 ± 0.06 | 4.89 ± 0.01; 7.60 ± 0.07; ≥9.31 ± 0.09 | 3.3 * | 4.0; 7.8 | ||
| ΔF/F (%) | Purified protein | 0 mM Mg | 103 ± 33 | 93 ± 11 | NA | NA | ||
| 1 mM Mg | 77 ± 16 | 78 ± 7 | ||||||
| HeLa cells | non-fluorescent | 169 ± 56 | ||||||
| Kd (nM) f | 0 mM Mg | 235 ± 37 (0.88 ± 0.12) | 289 ± 29 (1.54 ± 0.22) | |||||
| 1 mM Mg | 435 ± 39 (1.48 ± 0.18) | 466 ± 50 (1.54 ± 0.23) | ||||||
| Maturation half-time (min) | ND | ND | 28.2 ± 0.7 (sfGFP) 44 ± 2 (GAF-CaMP2) |
39 | 240 | |||
| Photobleaching half-time (s) g | ND | 1150 ± 560 | ND | 1080 ± 560 | 377 ± 55 * | ND | ||
a Data from [13]. Data marked with an asterisk were determined in this paper. NA, not applicable. ND, not determined. b Data from [12]. c QY for form with excitation maximum at 620−642 nm was determined at pH 7.20. miRFP720 was used as the reference standard [5]. d Extinction coefficient for form with absorbance maximum at 630−650 nm was determined relative to the Soret band at 384 nm for GAF-CaMP2 and relative to sfGFP form at 490 nm [29] for GAF-CaMP2-sfGFP. e Brightness was calculated as a product of the quantum yield and extinction coefficient and normalized to the brightness of EGFP that has an extinction coefficient of 56,000 M−1·cm−1 and quantum yield of 0.6 [30]. f Hill coefficient is shown in square brackets. In the absence and in the presence of 1 mM Mg2+ GCaMP6f had Kd values of 371 ± 20 nM (n = 2.4 ± 0.2) and 632 ± 15 nM (n = 2.25 ± 0.06), respectively. g The power of light from a mercury-xenon lamp before the objective lens was 2.23 mW/cm2.