ATPase/GTPase Activity Assay Kit Catalog Number MAK113 Storage Temperature 2–8 °C TECHNICAL BULLETIN Product Description ATPases and GTPases catalyze the decomposition of ATP or GTP into ADP or GDP and free phosphate. These enzymes play key roles in transport, signal transduction, protein biosynthesis, and cell differentiation. The ATPase/GTPase Activity Assay kit provides a simple and direct procedure for measuring ATPase/GTPase activity in a microplate format. This kit uses a single reagent formulation to accurately determine enzyme activity in 30 minutes at room temperature. The malachite green reagent forms a stable dark green color with free phosphate liberated by the enzymes resulting in a colorimetric product, measured at 620 nm (600–660 nm), proportional to the enzyme activity present. One unit is the amount of enzyme that catalyzes the production of 1 µmole of free phosphate per minute under the assay conditions. Components The kit is sufficient for 200 assays in 96 well plates. Reagent Catalog Number MAK113A 50 mL Phosphate Standard, 1 mM Catalog Number MAK113B 1 mL Assay Buffer Catalog Number MAK113C 10 mL Reagents and Equipment Required but Not Provided. • 96 well flat-bottom plate – It is recommended to use clear plates for colorimetric assays. • Spectrophotometric multiwell plate reader • ATP (Catalog Number A7699 or equivalent) and/or GTP (Catalog Number G8877 or equivalent) Precautions and Disclaimer This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. Storage/Stability This kit is shipped at room temperature. Storage at 2–8 °C, protected from light, is recommended. Preparation Instructions Briefly centrifuge vials before opening. Bring all reagents to room temperature prior to assay. Before each assay, it is important to check that enzyme preparations and assay buffers do not contain free phosphate. To test for free phosphate, add 200 µL of the Reagent to 40 µL sample solution. The blank absorbance values at 620 nm should be lower than 0.3. If the absorbance readings are higher, free phosphate may be a problem and phosphate levels should be checked. Lab detergents may be one source of phosphate contamination. Make sure labware is free from contaminating phosphate by rinsing thoroughly. Note: The provided Assay Buffer contains 40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, pH 7.5. Other buffers (HEPES, MES, MOPS), with the exception of phosphate buffers, can be used. Assay is compatible with 2 mM β-mercaptoethanol, 1 mM DTT, 0.5 mg/mL BSA, and 5% DMSO. Prepare a 4 mM solution of the ATP or GTP. Aliquot solution and store at –20 °C. Solutions should be used within 2 months of preparation. 2 Procedure Phosphate Standards Add 25 µL of the 1 mM Phosphate Standard to 475 µL of water to prepare 500 µL of a 50 µM phosphate standard solution. Number four tubes 1-4. Prepare standard solutions using 50 µM phosphate standard solution in numbered tubes as indicated in Table 1. Add 40 µL of appropriate standard to each well. Table 1. Phosphate Standards No Standard 1 2 3 4 200 µL 120 µL 60 µL 0 µL pmole Water Concentration (40 µL) 2,000 0 µL 50 µM 1,200 80 µL 30 µM 600 140 µL 15 µM 0 200 µL 0 µM Enzyme Activity Assay Reaction 1. Perform a series of dilutions of enzyme in assay buffer. Set up the sample reactions and the control well according to the scheme in Table 2. Table 2. Reaction Mixes Reagent Assay Buffer Enzyme sample Water 4 mM ATP or GTP Samples 20 µL 10 µL – 10 µL Control Well 20 µL – 10 µL 10 µL 2. Incubate the reaction for desired period of time (for example, 30 minutes) at room temperature. Optimal time may need to be determined experimentally. 3. Add 200 µL of Reagent to each well and incubate for an additional 30 minutes at room temperature to terminate the enzyme reaction and generate the colorimetric product. For best results, it is recommended to use a multichannel pipettor. 4. Read the absorbance at 600–660 nm [maximum absorbance at 620 nm (A620)]. Calculations Calculate the change in absorbance values (∆A620) for the samples by subtracting the A620 of the control well (A620)control from the A620 of the sample wells (A620)sample. Choose an enzyme measurement that gives a ∆A620 value between 0.5–1 to ensure the substrate hydrolysis (<10%) is within the linear kinetics of the reaction. Plot the standard curve and compute the concentration (µM) of free phosphate [Pi] in the sample from the standard curve. Enzyme activity = [Pi] (µM) × 40 µL (Units/L) 10 µL × T 40 µL = reaction volume 10 µL = sample volume T= reaction time One unit is the amount of enzyme that catalyzes the production of 1 µmole of free phosphate per minute under the assay conditions. Enzyme Inhibitor Assay Reaction 1. To evaluate an inhibitor or perform high-throughput screening, use the optimal enzyme concentration determined above and set up reactions according to the scheme in Table 3. Incubate enzyme and inhibitor together. The amount of time required for the inhibitor to inactivate the enzyme may need to be experimentally determined. Table 3. Inhibitor Assay Reaction Mixes Reagent Assay Buffer Enzyme sample Inhibitor DMSO Samples 20 µL 5 µL 5 µL – Control Well 20 µL – – 10 µL 2. Following this incubation, add the substrate (4 mM ATP or GTP) and incubate the reaction for the amount of time used in step 2 of the Enzyme Activity Assay Reaction. 3. Add 200 µL of Reagent to each well and incubate for an additional 30 minutes at room temperature to terminate the enzyme reaction and generate the colorimetric product. 3 Troubleshooting Guide Problem Possible Cause Cold assay buffer Omission of step in procedure Assay not working Plate reader at incorrect wavelength Type of 96 well plate used Samples used after multiple freeze-thaw Samples with erratic cycles readings Use of old or inappropriately stored samples Samples measured at incorrect Unanticipated results wavelength Samples contain interfering substances Suggested Solution Assay Buffer should be at room temperature Refer and follow Technical Bulletin precisely Check filter settings of instrument For colorimetric assays, use clear plates Aliquot and freeze samples if needed to use multiple times Use fresh samples and store correctly until use Check the equipment and filter settings If possible, dilute sample further LS,MAM 02/14-1 2014 Sigma-Aldrich Co. LLC. 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