Fura-2 Low Affinity (LowAff)

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Low affinity (formerly designated as “FF”) versions enable studies where a large concentration of calcium is expected. They have two major advantages over their high affinity counterparts. They cause reduced buffering of intracellular calcium and reduced perturbation of calcium transients, and they allow for measurements of shorter-lived transients.

TEFLabs low affinity calcium indicators, based on the effect of fluorines on lowering the affinity of the regular indicators, have an appropriate Kd in the range of 15 μM to 25 μM. Fura-2 LowAff and Indo-1 LowAff were the first low affinity indicators introduced by TEFLabs.

Fura-2 is also available in High Affinity (Fura-2), Near Membrane (Fura-2 NearMem), and Leakage Resistant (Fura-2 LeakRes) versions. TEFLabs offers the even lower affinity nitro versions of Fura-2 and Indo-1 on a custom synthesis basis.


Excitation: 340/380 nm (high/low Ca2+)

Emission:  505 nm

kd: 25 uM at 37 degrees C

Solubility: H20 (salt form)  DMSO (AM form)

Molecular Weight:  1038 g/mol (AM), 853 g/mol (K+salt)



FIG. 6.4 Fluorescence excitation traces (emission at 510 nm) of a Ca titration of Fura-2 LowAff

Fura-2 LowAff emission titration

The low affinity Ca2+ indicator selectivity over Mg2+ is illustrated in Figure 6.3.  Fura-2 (Figure 6.3a) is saturated at levels as low as 2 µM Ca2+.  This saturation may be avoided by using the higher Kd Furaptra or Fura-2 LowAff (Fura-2 FF). However, Furaptra (Figure 6.3b) cannot distinguish Mg2+ from Ca2+, such that it reports a high mitochondrial (M) concentration of Ca2+.  Fura-2 LowAff (Figure 6.3c) reports the correctly low mitochondrial concentration for Ca2+ because Mg2+ does not contribute to its fluorescence response.

FIG. 6.3

Fura-2 low Affinity




Fura-2 LeakRes

Fura-2 NearMem

Asante Calcium Green Low AFF

Asante Calcium Red LowAff

Fluo-2 lowAff