ATPase/GTPase Activity Assay Kit (MAK113) - Sigma-Aldrich

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The ATPase/GTPase Activity Assay kit provides a simple and direct procedure for measuring. ATPase/GTPase activity in a microplate format. This kit.
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. This high-sensitivity kit can detect ATPase and GTPase activity levels as low as 0.007 U/L. One unit is the amount of enzyme that catalyzes the production of 1 mole 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, Adenosine 5-triphosphate, (Catalog Number A7699 or equivalent) and/or GTP, Guanosine 5-triphosphate, (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 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. Use ultrapure water for the preparation of reagents and standards. 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 L of the Reagent to 40 L 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 -mercaptoethanol, 1 mM DTT, 0.5 mg/mL BSA, and 5% DMSO.

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Prepare a 4 mM solution of ATP or GTP in ultrapure water. Aliquot solution and store at –20 C. Solutions should be used within 2 months of preparation. Procedure All samples and standards should be run in duplicate. Phosphate Standards for Colorimetric Detection Dilute 25 L of the 1 mM Phosphate Standard with 475 L of ultrapure water to prepare 500 L of a 50 M Phosphate Standard Solution. Add 0, 10, 20, 25, 30, and 40 L of the 50 M Phosphate Standard Solution into a 96 well plate, generating 0 (blank), 500, 1,000, 1,250, 1,500, and 2,000 pmol/well (0, 12.5, 25, 31.25, 37.5, and 50 M) standards. Add ultrapure water to each well to bring the volume to 40 L. Sample Preparation Clear liquid samples can be assayed directly. Turbid samples or samples with precipitates should be clarified prior to use. Centrifuge at 14,000 rpm for 5 minutes to remove insoluble material. Transfer the supernatant to a second tube. Tissue samples (20 mg) or cells (1  10 cells) should be homogenized on ice with 200 L of ice cold Assay Buffer. Centrifuge at 14,000 rpm for 10 minutes to remove insoluble material and collect the supernatant. Transfer the supernatant to a second tube. 6

For unknown samples, it is suggested to test several sample volumes to make sure the readings are within the standard curve range. Add 1–10 L of samples into duplicate wells of a 96 well plate. Bring samples to a final volume of 10 L with Assay Buffer. Add 10 L of Assay Buffer into duplicate wells for a Negative Control. Note: High levels of phosphate can result in a sample background. To correct for this background, include a background blank for each sample. Add 200 L of Reagent to each background blank well immediately after adding the Reaction Mix to stop the reaction. Do not perform the initial 30 minute incubation in Step 3. The background blank readings can then be subtracted from the sample readings.

Assay Reaction 1. Set up the Reaction Mix according to the scheme in Table 1. 30 L of Reaction Mix is required for each sample, background blank, or negative control reaction (well). Table 1. Reaction Mixes Reagent Assay Buffer 4 mM ATP or GTP

Samples, Background Blanks, and Negative Controls 20 L 10 L

2. Add 30 L of Reaction Mix to each sample, background blank, and negative control well. Do not add to the standard wells. 3. Incubate the reaction for desired period of time (for example, 30 minutes) at room temperature. Optimal time may need to be determined experimentally. 4. Add 200 L 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. 5. Read the absorbance at 600–660 nm [maximum absorbance at 620 nm (A620)] for all samples, standards, blanks, and controls.

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Calculations Use the values obtained from the appropriate Phosphate Standards to plot a standard curve. Note: A new standard curve must be set up each time the assay is run. 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 0.5–1.0 to ensure the substrate hydrolysis (10%) is within the linear kinetics of the reaction. Note: It is essential that (A620) falls within the linear range of the standard curve. Using the A620 value, determine the concentration (M) of free phosphate in the sample well (Sa) from the standard curve. ATPase/GTPase Activity: Enzyme activity =

Sa  Rv Sv  T

Where: Sa = concentration of phosphate (M) generated in unknown sample well.

Sample Calculation: Amount of phosphate (Sa) = 35.84 micromolar (from standard curve) Reaction volume (Rv) = 40 L Sample volume (Sv) = 10 L added to well T= reaction time = 30 min ATPase/GTPase activity in sample well: mole/min/L =

35.84 M  40 L (30 min)  10 L

= 4.8 units/L

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 2. Incubate enzyme and inhibitor together. The amount of time required for the inhibitor to inactivate the enzyme may need to be experimentally determined. Table 2. Inhibitor Assay Reaction Mixes Reagent Assay Buffer Enzyme sample Inhibitor Buffer/DMSO

Samples 20 L 5 L 5 L –

Control Well 20 L – – 10 L

Rv = reaction volume (L) Sv = sample volume (L) added to well T=

reaction time (min)

One unit is the amount of enzyme (ATPase or GTPase) that catalyzes the production of 1 mole of free phosphate per minute under the assay conditions.

2. Following this incubation, add the substrate (4 mM ATP or GTP) and incubate the reaction for the amount of time used in Step 3 of the Enzyme Activity Assay Reaction. 3. Continue the assay from Step 4 of the Enzyme Activity Assay Reaction.

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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 cycles Samples with erratic 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 KVG,MF,LS,MAM 08/16-1

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