Pfk activity colorimetric assay kit

Measurement of PFKFB3 activity was performed by Phosphofructokinase (PFK) Activity Colorimetric Assay Kit (Sigma). Briefly, purified WT or mutant proteins were added to the PFK Assay Buffer, supplemented with PFK Enzyme Mix, PFK Developer, ATP and PFK Substrate. The blank was setted without PFK Substrate. PFK activity is determined by a coupled enzyme assay, in which fructose-6-phosphate and ATP is converted to fructose-1,6-diphosphate and ADP by PFK. The ADP is converted by the enzyme mix to AMP and NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the PFK activity present. One unit of PFK is the amount of enzyme that will generate 1.0 μmole of NADH per minute at pH 7.4 at 37 °C. PFK activity is reported as nmole/min/mL = milliunit/mL. A standard curve was conducted with diluted NADH according to the kit. Incubate the plate at 37 °C and measure the absorbance at 450 nm every 5 min. The final measurement would be the penultimate reading. The enzyme activity was calculated according to the equation provided by the kit.

PFK Activity Colorimetric Assay Kit was applied to evaluate the activity of phosphofructokinase (PFK) (Sigma-Aldrich, USA). Treated cells were mixed with PFK Assay Buffer and under cell lysis with Reaction Mix according to the manufacturer's instructions. Microplate reader was used to test the OD value of the mixtures per 30 s. One unit of PFK mediates 1.0 μM per minute of NADH generation. A standard line of NADH was built for PFK activity calculation. After normalization to the protein concentration, the PFK activity was showed as milliunits/mg of protein.

The activity of phosphofructokinase (PFK) was measured using the PFK Activity Colorimetric Assay Kit (Sigma-Aldrich, St. Louis, MO, USA), by which PFK-mediated NADH generation in the reaction mix is reflected by the OD change at 450 nm. Treated cells were homogenized with PFK Assay Buffer. Cell lysis was performed using the Reaction Mix prepared according to the manufacturer's instructions. The OD of the mixtures was measured per 30 s with the microplate reader. A NADH standard was also established for PFK activity calculation. One unit of PFK is the amount of enzyme that generates 1.0 μM of NADH per minute. After normalization of the protein concentration, the PFK activity was expressed as milliunits/mg of protein.