This protocol will aid in the determination of the activity for ATPase proteins by measuring the free phosphate resulting from ATP hydrolysis by the proteins. The malachite green assay is a simple, sensitive, fast, and inexpensive procedure for the detection of inorganic phosphate. It is suitable for automation and high-throughput screening of compounds against its target.
This technique will play an important role in the rapid discovery of inhibitors against the Hsp90 protein, which is an important anti-cancer target. To begin, pipette 40 microliters of one-millimolar phosphate standard in 960 microliters of ultrapure water to obtain 40-micromolar phosphate solution. Add this solution as per the manufacturer's instructions to form a serial dilution of phosphate from 0-to 40-micromolar.
Next, use eight microliters of yeast Hsp90 for measurable ATPase activity. Add malachite green reagent to the blank and the positive control, and check the the optical density difference between the blank and the positive control after the addition is at least 0.5 and less than 1. To prepare ATP, transfer one milligram of ATP to a vial containing 453.39 microliters of ultrapure water to obtain four-millimolar stock solution.
Prepare the ATP fresh, as it can spontaneously hydrolyze over time. Add four microliters of ATP stock solution to each well to give a final well concentration of 0.2-millimolar. Add ATP as the last component to the reaction using a multi-channel pipette so that all the reactions start simultaneously.
Next, prepare a 10-millimolar stock of geldanamycin by dissolving one milligram in 178.37 microliters of DMSO. Using the stock solution, prepare 4-millimolar, 0.8 millimolar, 0.16-millimolar, 0.032-millimolar, 0.0064-millimolar, and 0.00128-millimolar solution in DMSO by serial dilution. Add two microliters of these stock solutions to each well to obtain a final well concentration of 0.1-millimolar, 0.02-millimolar, 0.04-millimolar, 0.0008-millimolar, 0.00016-millimolar, and 0.000032-millimolar.
Then, prepare the wells along with the wells containing the blank and negative control. The wells containing geldanamycin serve as the positive control. Prepare the 96-well plates and prepare a separate well for the compounds that show absorbance at 620 nanometers to record the absorbance at this wavelength.
Do not show a separate well for geldanamycin, as geldanamycin does not show absorbance at 620 nanometers for the concentration used in this assay. Set up assay wells by adding ultrapure water to each well. Then, add the required amount of assay buffer and a compound solution such as geldanamycin solution.
Add Hsp90 to the appropriate wells and shake the plates for two minutes on a plate shaker at room temperature. Add four microliters of ATP solution using a multi-channel pipette. Wrap the plate in aluminum foil and shake for two minutes on a plate shaker at room temperature at 200 rpm.
Incubate the plate at 37 degrees Celsius for three hours. Stop the reaction by adding 20 microliters of malachite green reagent in the same order as ATP using a multi-channel pipette and incubate for 15 minutes at room temperature. Finally, measure the absorbance of the plate at 620 nanometers in a plate reader.
In addition plate A, add 90 microliters of assay buffer in each well. In addition plate B, add 90 microliters of buffer with Hsp90 in each well. In addition plate C, add 90 microliters of ATP, and in addition plate D add 90 microliters of malachite green reagent in each well.
Use an automated multi-channel dispensing system to transfer 40 microliters of solution from each well of addition plate A to main plates one and two, and plate B to main plate three and four. Transfer 20 microliters of the solution from each well of the compound plate to main plates one, main plates two, main plates three, and main plates four. Shake the plates for one minute in a shaker at 200 rpm.
Transfer 20 microliters of the solution from each well of the addition plates C to main plates one, two, three, and four. Shake the plates for one minute in a shaker at 200 rpm. Incubate the plates at 37 degrees Celsius for three hours and then transfer 20 microliters of malachite green reagent from addition plate D to the wells of main plates one, two, three, and four.
After a 15-minute incubation at room temperature, measure the absorbance of the plate at 620 nanometers in a plate reader. The structure of malachite green and the reactions of malachite green reagent A with free phosphate and the subsequent reaction with reagent B are shown here. A standard plot for standard phosphate concentration is represented in this figure.
Here, the x-axis represents the free phosphate or pi concentration in micromolar, and the absorbance at 620 nanometers is depicted on the y-axis. The error bars represent the deviation between the two sample numbers. The percentage ATPase activity is used to measure the dose-dependent inhibition of the protein by geldanamycin.
It was observed that geldanamycin inhibited the protein in a dose-dependent manner with an IC50 value of 0.85-micromolar. Here, each point is a mean of two determinations. The color development in main plate one after the addition of the malachite green agent is shown in this image.
The pink color in the A11 well is due to the compound color. Similarly, the color development in main plate two is shown here. The pink color in the A11 well is due to the colored nature of the compound.
The color development after the addition of the malachite green reagent in main plate three and in main plate four is shown here. The addition of ATP and malachite green reagent at the same time interval to every well is an important thing to remember when attempting this procedure.
