Sigma aldolase kit

The enzyme activity of TciALDO was measured at 30 o C in a coupled assay with reversible conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate using Sigma aldolase kit (Catalogue # MAK223). The NADH production was measured colorimetrically at 450 nm. The final reaction mixture (100 ul) contained assay buffer, enzyme mix, enzyme developer, recombinant protein (50 µg) and the substrate. NADH standards and the blank were set up as described by the manufacturer.
(1) The optimum pH was determined with a substrate concentration of 0.5 mM fructose 1,6-bisphosphate with pH range 6 to 9. Subsequent assays were carried out at pH 7.5.
(2) The apparent K m for fructose 1,6-bisphosphate was determined in reaction mixtures containing 0-5 mM fructose 1,6-bisphosphate.
(3) The effects of EDTA as potential activators/inhibitors on recombinant TciALDO with substrate concentrations of 0.5 mM fructose 1,6-bisphosphate and 10 mM EDTA.

The enzyme activity of TciALDO-1 was measured at 30 • C in a coupled assay with reversible conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate using a Sigma aldolase kit (Catalogue # MAK223, St. Louis, MO, USA). NADH production was measured colorimetrically at 450 nm. The final reaction mixture (100 µL) contained assay buffer, enzyme mix, enzyme developer, recombinant protein (50 µg), and the substrate. NADH standards and the blank were set up as described by the manufacturer.
(1) The optimum pH was determined (in three independent biological replicates) with a substrate concentration of 0.5 mM fructose 1,6-bisphosphate with a pH range of 6 to 9. Subsequent assays were carried out at pH 7.5.
(2) The apparent K m for fructose 1,6-bisphosphate was determined (in three independent biological replicates) in reaction mixtures containing 0-5 mM fructose 1,6-bisphosphate.
(3) The effects of EDTA as potential activators/inhibitors on recombinant TciALDO-1 with substrate concentrations of 0.5 mM fructose 1,6-bisphosphate and 10 mM EDTA were measured.