Table 1. Cysteine-reactive probes used in VCF studies
ProbeSpectral propertiesUsage notesExample references
Monobromobimaneλex = 394 nmVery small size compared with other probes allows labeling of less accessible locations with lower risk of structural perturbation. Near-UV excitation may cause aberrant photodamage.Islas and Zagotta, 2006; Taraska et al., 2009
λem = 490 nm
DPTA-Tb3+λex = 328 nmTerbium chelate used in LRET studies. Reduced orientation-dependence of energy transfer makes it a more reliable reporter for distance changes than conventional fluorophores. Requires laser excitation in UV range.Cha et al., 1999; Posson et al., 2005
λem = 492 or 546 nm
Fluoresceinλex = 494 nmBright, environmentally sensitive fluorescent probe but has pH sensitivity in the physiological range (pKa = 6.4) and is prone to photobleaching.Cha and Bezanilla, 1997; Dempski et al., 2006
λem = 512 nm
Alexa Fluor 488 C5λex = 493 nmBright fluorescent probe with low pH sensitivity in the physiological range. Lower environmental sensitivity of emission makes it less sensitive to conformational changes in absence of added quenchers.Zheng and Zagotta, 2000; Bruening-Wright et al., 2007
λem = 516 nm
Oregon Greenλex = 501 nmDerivative of fluorescein with reduced pH sensitivity due to lower pKa (4.6) and lower rate of photobleaching. Emission is highly sensitive to calcium concentrations.Cha and Bezanilla, 1997
λem = 526 nm
PyMPOλex = 415 nmEnvironmentally sensitive probe whose linear shape makes it less bulky than alternatives such as fluorescein or tetramethyl rhodamine. Extended shape means probe reaches far from point of labeling.Savalli et al., 2006; Vaid et al., 2008
λem = 570 nm
Tetramethyl rhodamineλex = 548 nmMost commonly used probe with high environmental sensitivity and good photostability.Mannuzzu et al., 1996; Cha and Bezanilla, 1997
λem = 576 nm

DPTA, diethylenetriaminepentaacetic acid; PyMPO, 1-[3-(succinimidyloxycarbonyl)benzyl]-4-[5-(4-methoxyphenyl)-2-oxazolyl]pyridinium bromide.