am performing a Threonine Synthase (TS) activity assay using a malachite green–ammonium molybdate-based phosphate detection method at 660 nm. The assay conditions include 100 mM TAPS buffer (pH 8.4), 0.1 mM PLP, 4 mM OPH (fixed substrate concentration), and varying enzyme concentrations range (2–60µg, 0.1 mL total volume at 25°C).
Ideally, I expect a typical enzyme saturation curve, but my data show unexpected fluctuations—a mid-range dip followed by an increase at higher enzyme concentrations. This pattern is inconsistent . Similar trends are observed at different reaction times (15, 30, and 45 min).
I would greatly appreciate any insights or suggestions from the community regarding potential causes for this inconsistency and how to resolve it. If anyone has encountered similar issues or can recommend references or protocols, I would be very grateful.
Best regards,
Mohd Javed
I would say that the enzyme concentrations are too high. I suggest you use 2 micrograms as the highest amount of enzyme, and collect more time points, evenly spaced. Then you can plot progress curves (product concentration versus time) for each enzyme concentration. These should have a linear portion (initial rate) starting from the beginning of the reactions. Measure the slope of the linear portion. Plot the slope of each progress curve versus the enzyme concentration. This plot should be linear, indicating that the rate is proportional to the enzyme concentration.
Also prepare a standard curve of absorbance versus phosphate concentration, which should be used to convert the absorbance to the phosphate concentration. There is a limit to the linearity of this relationship, so a standard curve is needed to make the conversion accurately.
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