Hi. I am using a malachite green assay to measure phosphate production for ATPase activity (MAK113 Sigma ATPase/ GTPase Activity Assay). In the protocol it says to subtract the absorbance (measured at 620) of the control (10 ul Assay Buffer + 30 ul reaction mixture (20 ul Assay buffer + 10 ul 4mM ATP) + 200 ul Reagent) from the absorbance of the samples. You then perform a calculation to get the enzyme activity. However is it not more accurate to calculate the phosphate production of the control (there is always a tiny amount) and subtract this from the samples? If the latter is done you get a diferent activity. To me this seems more logical as you account for the phosphate in the control compared to just the difference in absorbance.
It also says to incubate at room temperature but wouldn't you obtain a more realistic result if you incubate the assay at physiological temperature?
Thank you for any answers or advice.
My preference would be to subtract the absorbance of a negative control sample that either lacked the enzyme, had a completely inhibited enzyme, or lacked the non-ATP substrate. This control would be subjected to the same reaction conditions as the active samples. This is important because some of the ATP may break down during the reaction and release phosphate, in addition to the phosphate contamination that was in the ATP at the start.
The standard curve of absorbance versus phosphate concentration has a limited linear range. If the phosphate concentration is beyond this range, a simple background absorbance subtraction will be inaccurate. In such a case, the absorbance values should be converted to phosphate concentrations before subtracting the background.
The Malachite Green phosphate detection reagent is used for endpoint measurements because it is strongly acidic and will quench the enzyme reaction. Once it is added, there is no point in continuing the incubation at physiological temperature. Doing so would just increase the amount of phosphate released by the spontaneous breakdown of ATP.
Dear Adam,
as I understand it, Stephen is performing the control you describe (or at least the manual says so).
IMHO there is no difference between first substracting and then converting to P concentration and first converting and then substracting. That's only mathematical procedure.
But of course one has to check, whether the absorbance in the sample is in the range in first place.
Hi Tomáš,
The difference can arise if one fails to take into account the nonlinearity of absorbance versus phosphate concentration beyond a certain limit, and instead assumes that the absorbance is always directly proportional to phosphate concentration. If one never exceeds the linear range of the standard curve, then there is no difference.
I have also seen with one commercial Malachite Green reagent a lag in detection at the low end of the standard curve. This could lead to a significant error of the same type when working near the bottom end of the standard curve.
I agree with the answers of Dr. Shapiro. We do controls for non-enzymatic hydrolysis of ATP (or ADP or AMP) by incubating the substrates in the same conditions of our test samples but without the addition of the enzyme (or in absence of cells). Then, after we stop the reaction with TCA we add the substrate and measure the Pi released by malachite green.
In relation to the standard curves we also note that the points of Pi concentrations below 2 nmol we have a "desviation" of the "Lambert and Beer factor".
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