Live-Cell Cytokinin-Binding Assay

Intact E. coli cultures (strain KMI001), expressing CRE1/AHK4 cytokinin receptor (Suzuki et al., 2001; Yamada et al., 2001), were grown at 25°C overnight. M9 liquid medium, supplemented with casamino acids [0.1% (w/v)] and ampicillin (100 μg/ml), were used to reach OD₆₀₀ ∼1–1,4. The assay described by Romanov et al. (2005) was performed with slight modifications.

Each sample contained 1 ml of the overnight cell culture, 3 pmol of [³H]tZ and various concentrations of unlabeled tZ/other tested compound (0.1 nM–50 μM). Negative control contained 3 pmol of [³H]tZ and 0.1% (v/v) dimethylsulfoxide (DMSO; solvent), instead of the unlabeled compound. After 30 min incubation at 4°C, the sample was centrifuged (8,000 rpm, 4 min, 4°C) and supernatant was removed. Bacterial pellet was resuspended in 50 μl dH₂O. Subsequently, 1 ml of scintillation cocktail was added. Radioactivity was measured by a Hidex 300 SL scintillation counter Hidex (FL). High excess of unlabeled tZ (at least 3000-fold) was used for competition, to discriminate between specific and non-specific binding.