Zinc assay kit

Following kits were used for determination of concentrations of calcium, iron, and zinc ions: Calcium Assay Kit (Colorimetric) (Abcam, ab102505), Iron Assay Kit (Colorimetric) (Abcam, ab83366), and Zinc Assay Kit (Abcam, ab102507), respectively. In cellular analyses, 6 × 10⁵ cells were passaged in 10 cm diameter-plates and allowed to attach overnight. Cells were then harvested and transferred to tubes, washed with the PBS buffer (137 mM NaCl; 2.7 mM KCl; 10 mM Na₂HPO₄; 1.8 mM KH₂PO₄), and centrifuged (300 x g for 10 min). The experiment was performed in 3 independent replications. In animals tissues analysis, 10 mg of brain, liver or spleen were separated and washed with the PBS buffer. Cellular pellets and tissues were next lysed/homogenized for 3 h with Calcium Assay Buffer, Iron Assay Buffer or EDTA-free lysis buffer (0.9% NaCl; 0.5% Triton X-100; 0.1% SDS; 1% sodium deoxycholate; 50 mM Tris-HCl pH 7.5) to determine concentrations of calcium, iron or zinc ions, respectively. To obtain cellular lysate or tissue homogenate, samples were centrifuged at 16,000 x g for 10 min. Calcium, iron or zinc ion concentrations were assessed by colorimetric measurements and appropriate calculations, according to the manufacturer’s instructions.

The zinc concentration of TrcP in solution was measured with a Zinc Assay Kit (Abcam) according to the manufacturer’s instruction. Twenty-five μl of TrcP solution (97.6 μM) was mixed with 25 μl 14% trichloroacetic acid or water, followed by centrifugation and recovery of the supernatant. A 50 μl serial dilution series of a Zn standard solution (0–100 μM) and TrcP samples were mixed with a 200 μl reaction mix and incubated for 10 min at room temperature. Light absorption at A₅₆₀ was measured. Zinc concentrations of TrcP solutions were calculated with a standard curve generated with the standard Zn solutions.