Jul 29, 2025
Detection of BLM Helicase ATPase Activity with the Transcreener® ADP² Kinase Assay
This protocol is a draft, published without a DOI.
- BellBrook Labs1
- 1Madison, WI
- BellBrook Labs Protocols
Protocol Citation: BellBrook Labs 2025. Detection of BLM Helicase ATPase Activity with the Transcreener® ADP² Kinase Assay. protocols.io https://protocols.io/view/detection-of-blm-helicase-atpase-activity-with-the-g6igbzcbx
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 29, 2025
Last Modified: July 29, 2025
Protocol Integer ID: 223528
Keywords: detection of blm helicase atpase activity, blm helicase atpase activity, dependent atpase activity of blm, transcreener adp² kinase assay, use with purified blm helicase, purified blm helicase, dependent atpase activity, mutations in blm, fluorescence intensity, atp hydrolysis, assay, inhibitor dose response measurement, enzyme, bloom syndrome, competitive fluorescence polarization, family helicase
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Abstract
Bloom syndrome helicase (BLM) is a RecQ-family helicase that uses the energy released by ATP hydrolysis to remodel aberrant DNA structures. Mutations in BLM are associated with Bloom syndrome, which causes premature aging and cancer predisposition. This application note describes methods for using the Transcreener ADP² Kinase Assay to measure the DNA-dependent ATPase activity of BLM. ADP produced by the enzyme is directly detected by a far-red, competitive fluorescence polarization (FP), Fluorescence intensity (FI), or time-resolved Förster-resonance-energy-transfer (TR-FRET) assay. The assay has been optimized and extensively validated for high-throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers. The assay is designed for use with purified BLM Helicase; it is not intended for use with cells or cellular lysates.
Materials
Component | Notes
--- | ---
Transcreener ADP² Assay | Transcreener ADP² FP Assay (Part# 3010)
Transcreener ADP² FI Assay (Part# 3013)
Transcreener ADP² TR-FRET Assay (Part# 3011)
ATP and ADP | 5 mM in Nuclease Free Water. Included in the Transcreener kit.
BLM Helicase Enzyme | Recombinant human BLM (amino acids 630–1290), C-terminal His tag, 76.2 kDa (Part# 2353 and 2354).
Sheared salmon sperm DNA | 10 mg/mL in Nuclease Free Water (Part# 2324 and 2325); to be used at 1 µg/mL in the BLM reaction.
Assay Buffer | 1X Enzyme Assay Buffer H (Part# 2349 and 2350) with 1 mM DTT (Part #2296 and 2297). Final buffer components are 50 mM Tris (pH 8.0), 5 mM MgCl₂, 100 mM NaCl, 0.01% Triton X-100, and 1 mM DTT.
Assay Plates | FP and FI Assay: Corning 384-Well Black Assay Plates (Part# 4514)
TR-FRET Assay: Corning 384-Well White Assay Plates (Part# 4513)
Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Use nuclease free water such as Invitrogen Cat. #AM9930
Liquid Handling Devices | Liquid Handling Devices that can accurately dispense sub-microliter volumes into 384-well plates.
Plate Reader | [List of compatible plate readers and settings.](https://www.bellbrooklabs.com/)
Troubleshooting
Introduction
Bloom syndrome helicase (BLM) is a RecQ-family helicase that uses the energy released by ATP hydrolysis to remodel aberrant DNA structures. Mutations in BLM are associated with Bloom syndrome, which causes premature aging and cancer predisposition. This application note describes methods for using the Transcreener ADP² Kinase Assay to measure the DNA-dependent ATPase activity of BLM. ADP produced by the enzyme is directly detected by a far-red, competitive fluorescence polarization (FP), Fluorescence intensity (FI), or time-resolved Förster-resonance-energy-transfer (TR-FRET) assay. The assay has been optimized and extensively validated for high-throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers. The assay is designed for use with purified BLM Helicase; it is not intended for use with cells or cellular lysates.
Figure 1. Schematic Overview of the Transcreener ADP² FP, FI, and TR-FRET Assay. ADP produced by the target enzyme displaces a tracer from the ADP² antibody, resulting in decreased fluorescence polarization for FP assay, decreased TR-FRET for TR-FRET assay, and increased fluorescent intensity for FI assay.
Methods
We describe three common procedures for enzymatic assays in a drug discovery setting. Depending on your goals, it may not be necessary to perform every procedure.
Performing an enzyme titration to determine the appropriate concentration for a good assay signal.
Generating a standard curve to enable conversion of the assay signal to amount of product formed, necessary for accurate IC50 measurements.
Generating a dose response curve for an inhibitor and determining the IC50.
The methods described here are for endpoint detection of BLM activity under initial velocity conditions (≤ 10% substrate conversion) with a K_m concentration of ATP (100 µM) and a saturating concentration of Sheared salmon sperm DNA (1 µg/mL). These conditions will ensure sensitive detection of inhibitors that compete with ATP. The assay protocol is for a 384-well format, using a 10 µL enzyme reaction and 10 µL ADP Detection Mix. The use of different formats will require changes in reagent quantities (see Transcreener ADP² Tech manual).
Simple Mix-and-Read Format. The enzyme reaction is incubated for 60 min followed by addition of 1X ADP Detection Mix, which contains EDTA to quench the reaction. Plates are allowed to sit for 60 min at room temperature to allow the fluorescent signal to reach equilibrium.
Figure 3. Sample Data for FP Readout Mode. a) Enzyme titration curve. b) Raw polarization in enzyme titration curve is converted to ADP formed using a standard curve. Only the linear portion of the graph is shown; interpolation was performed using GraphPad Prism. c) Dose-response curve of the probe inhibitor, Suramin. d) Z’ measurement (n=16).
Performing a BLM Enzyme Titration
Using the EC_80 concentration suggested in the BLM Enzyme Certificate of Analysis should provide a robust signal that is within the linear range for ADP formation. However, for best results, it may be useful to perform an enzyme titration to identify the optimal enzyme concentration. An enzyme concentration that produces 80% of the maximal signal will typically meet these criteria (see Figure 3a).
Prepare Working Stocks
Prepare Assay Buffer containing 1X Enzyme Assay Buffer H and 1 mM DTT in Ultrapure Nuclease-Free Water.
Dilute enzyme to 2X the desired concentration (20 nM in the example shown in Figure 3a) in Assay Buffer.
Prepare 2X Substrate Mix containing 200 µM ATP and 2 µg/mL salmon sperm DNA in Assay Buffer.
Perform Enzyme Titration and Enzyme Reaction
Add 5 µL of Assay Buffer to well 2-16.
Add 10 µL of 2X enzyme to well 1. Perform a two-fold serial dilution by pipetting 5 µL of the 2X enzyme solution from well 1 to well 2, and so on to well 15. Well 16 should be kept as a No Enzyme control.
Add 5 µL of 2X Substrate Mix to each well to initiate the reaction. Mix gently on a plate shaker and incubate at 30°C for 60 minutes.
Prepare and Dispense ADP Detection Mix
Centrifuge ADP Antibody at 10,000 x g for 10 minutes to remove any precipitate. It is normal for some precipitation to occur over time or after freeze/thaw cycles; removing it will not affect assay performance.
Prepare 1X ADP Detection Mix by diluting Stop 6 Detect Buffer, ADP Tracer, and ADP Antibody in Ultrapure Nuclease-Free Water to the concentrations described in Table 1, according to the appropriate readout mode.
Add 10 µL of the 1X ADP Detection Mix to each well and mix gently on a plate shaker. Incubate at room temperature for 60 minutes to allow the detection reaction to reach equilibrium.
Table 1. 1X ADP Detection Mix Components. The optimal concentrations for each of the detection reagents based on the preferred readout mode are shown. Significant changes in the ATP concentration may require adjustment of the concentration of ADP Antibody (FP and FI Assay) or ADP Tracer (TR-FRET Assay) as described in the Transcreener ADP² Tech manual.
Generating an ATP/ADP Standard Curve
To quantify enzyme activity or obtain an accurate inhibitor IC50, a standard curve is used to convert the raw signal into the amount of ADP produced. This standard curve mimics the enzyme's conversion of ATP to ADP. Starting with the initial ATP concentration, ATP is sequentially decreased while ADP is increased proportionally (see Figure 4). Data interpolation can be performed using GraphPad Prism.
Prepare Assay Buffer as described in Method 1.
Dilute ATP and ADP to 100 µM in Assay Buffer. Then add proportional quantities of ATP and ADP in separate microcentrifuge tubes to create ATP/ADP mix as in the table in Figure 4. For example, combine 60 µL of 100 µM ATP and 40 µL of 100 µM ADP to generate an ATP/ADP mix that mimics 40% substrate conversion.
Add 10 µL of each ATP/ADP mix to a separate well.
Prepare and dispense ADP Detection Mix as described in Method 1.
Generating an Inhibitor Dose Response Curve
For Single Compound Screening and Dose-Response Assays, following the following protocol:
Prepare Working Stocks and Dispense Test Compounds
Prepare Assay Buffer and 2X Substrate Mix as described in Method 1.
Dilute enzyme to 2X the desired concentration, typically 2X EC80 of the enzyme titration in Method 1, in Assay Buffer.
Dispense test compounds into wells as a concentration series for the Dose Response Curve (DRC). Typically, 12 concentrations generated by two-fold serial dilution from 100-fold the expected IC50 are sufficient for the DRC. The final concentrations of test compounds should be based on the 10 µL fi
nal volume of the enzyme reaction.
Perform Enzyme Reaction
Add 5 µL of 2X enzyme to each well. Incubate for 30 minutes at room temperature to allow equilibration of the E-I complex.
After the incubation, add 5 µL of 2X Substrate Mix to each well to initiate the reaction. Mix gently on a plate shaker and incubate at 30°C for 60 minutes.
Prepare and Dispense ADP Detection Mix as described in Method 1.
To calculate the IC50, use the standard curve (described in Method 2) to interpolate the quantity of product generated at each concentration of inhibitor, then convert to % Inhibition. More information on this process can be found here.
Acknowledgements
Ordering Information
Please visit [www.bellbrooklabs.com](http://www.bellbrooklabs.com) or contact BellBrook Labs for assay pricing. Custom quotes are available for bulk orders.
Email Orders: [email protected]
Phone Orders: (608) 443-2400
Toll-Free: (866) 313-7881
Fax Orders: (608) 441-2967
Technical Information
For Technical Information, please contact one of our BellBrook scientists:
Email: [email protected]
Telephone: (608) 443-2400
Toll-Free: (866) 313-7881
Additional Ordering and Technical Information:
Ordering Information
Please visit www.bellbrooklabs.com or contact BellBrook Labs for assay pricing. Custom quotes are available for bulk orders.
Email Orders: [email protected]
Phone Orders: (608) 443-2400
Toll-Free: (866) 313-7881
Fax Orders: (608) 441-2967
Technical Information
For Technical Information, please contact one of our BellBrook scientists:
Email: [email protected]
Telephone: (608) 443-2400
Toll-Free: (866) 313-7881