4 mm atp

The ATPase activity was evaluated by the measurement of the colorimetric product resulting from the malachite green reagent and free ion [PO4]³⁻ measured at 620 nm. The ATPase assays were carried out using an ATPase/GTPase Activity Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). The assay reaction mixture was composed of 10 µg recombinant protein (for the ATPase activity measurements) incubated in a 30 uL reaction volume containing 20 µL 40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA at pH 7.5, and 10 uL 4 mM ATP (Sigma-Aldrich, St. Louis, MO, USA). To monitor the effect of temperature on the activity of PCBG5HP1, PCBG5HP2, and PCBG5HP12, the ATPase reaction mixture was incubated at room temperature or 4 °C for 30 min. After incubation, the reaction was stopped by adding 200 µL of malachite green reagent and incubated for an additional 30 min at room temperature to generate the colorimetric product. The product mixtures were loaded onto a 96-well plate and the absorbance values of colorimetric products were read using a SpectraMax spectrophotometer (Molecular Devices, San Jose, CA, USA) at 620 nm. All samples were run in triplicates. Phosphate standard values for colorimetric detection were prepared according to the manufacturer’s instructions.

The ATPase activity was evaluated by the measurement of colorimetric product resulting from the malachite green reagent and free ion [PO₄]³⁻ measured at 620 nm. The ATPase assays were carried out using an ATPase/GTPase Activity Assay Kit (Sigma-Aldrich, USA). The assay reaction mixture was composed of 10 µg recombinant protein (for the ATPase activity measurements) incubated in a 30 uL reaction volume containing 20 µL 40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA at pH 7.5, and 10 uL 4 mM ATP (Sigma-Aldrich, USA). To monitor the effect of temperature on the activity of GaHSP70-1 and GaHSP70-2, the ATPase reaction mixture was incubated at 37, 25, 15, and 4 °C for 1 h. After 1 h of incubation, the reaction was stopped by adding 200 µL of malachite green reagent and incubated for an additional 30 min at room temperature to generate the colorimetric product. The product mixtures were loaded onto a 96-well plate and the absorbance values of colorimetric products were read using a SpectraMax spectrophotometer (Molecular Devices, San Jose, CA, USA) at 620 nm. All the samples were run in triplicate. Phosphate standard values for colorimetric detection were prepared according to the manufacturer’s instructions.