Table 1. Summary of GST-Rab7 binding to BODIPY nucleotide analogs.

Experiments carried out in 1 mM EDTA buffer unless specified

Nucleotide	BODIPY FL GDP	BODIPY FL GTP
1Kd (nM)	54 ± 15	150 ± 62
2Bmax (103 BODIPY/bead)	2000 ± 400	1600 ± 400
3t1/2 (mins)		11.1 ± 0.6
4koff (sec−1)	2.3 ± 1.5 × 10−3	7.0 ±3.0 × 10−3
4koff (sec−1) Mg2+		1.3 × 10−3

Competitors	GDP	GTP-γ-S
5Ki (nM)	13 ± 5	40 ± 10

^1,2Maximal number of bound BODIPY nucleotide molecules per bead ( B_max ) and dissociation constants were calculated simultaneously using the GraphPad Prism software to fit dose dependence to $B=B_{\text{max}}\frac{\left[\mathit{\text{Ligand}}\right]}{K_d+\left[\mathit{\text{Ligand}}\right]}$ where B represents the number of bound receptors. Cytometry detector output was converted to BODIPY/bead using standard fluorescein beads from Invitrogen.

³Observed on-rate was calculated using B = B_max(1−e^{−k_obs(time)}) to fit the kinetic data.

⁴Off rates were calculated by fitting B = Span(e ^{−k_off(time)}) + Plateau to fit kinetic data.

⁵Competition of non-fluorescent nucleotide and BODIPY FL GTP. Experimental data was fit to $\mathrm{B}={\mathrm{B}}_{\text{min}}+\frac{\mathit{\text{Span}}}{1+{10}^{\left(\left[\mathit{\text{Ligand}}\right]-\text{log}{\mathit{\text{EC}}}_{50}\right)}}.$ Inhibition constants were calculated by converting the fit EC₅₀ values to $K_i=\frac{{\mathit{\text{EC}}}_{50}}{1+\frac{100\mathit{\text{nM}}}{K_d}}.$