Transcreener ADP² Assay directly measures the ADP produced by BLM Helicase (Bloom Syndrome Helicase). The ADP measurements allow researchers to effectively determine the activity of the BLM Helicase. The assay provides a powerful tool for screening compound libraries for BLM Helicase modulators to help find new therapies for disease.
The kit includes the detection reagents required to measure activity. The BLM Helicase enzyme is not included in the inhibitor screening assay kit but can be purchased through BellBrook Labs.
Looking for a detailed protocol for performing the assay in your lab? Download the application note to get the assay up and running in your lab within hours.
Learn how Trancreener’s flexibility can save you time and resources.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The BLM Helicase activity assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized BLM Helicase reaction conditions indicate a robust assay. Robust data like this is vital for sizeable, high-throughput screens that are difficult to complete due to the massive sample quantity. Z’ shown here = 0.83.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here, we used 50 mM Tris (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 0.01% Triton X-100, and 1 mM DTT for the buffer, and ran with 1 µg/mL sssDNA and 100 µM ATP at 30°C for 1 hour. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. Although sheared salmon sperm DNA was used for the displayed data, other DNA substrates may be used; however, it is important to note that the DNA may need to be titrated for the best result.
Transcreener Assays are designed to screen compound libraries in a high-throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease.
Assay conditions for this dose-response assay include 50 mM Tris (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 0.01% Triton X-100, and 1 mM DTT, and ran with 1 µg/mL sssDNA, and 100 µM ATP at 30°C for 1 hour.
Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.
Interested in moving your program forward but don’t want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your BLM Helicase program.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| Stop & Detect Buffer B, 10X for FP & FI, Stop & Detect Buffer C, 10X for TR-FRET | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35. Stops enzyme reactions requiring Mg²+. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. |
| Component | Notes |
|---|---|
| BLM Helicase Enzyme | We have successfully used recombinant human BLM Helicase (aa 630-1290) expressed and purified from Baculovirus infected insect cells (BellBrook Labs). |
| Assay Buffer | 50 mM Tris (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 0.01% Triton X-100, and 1 mM DTT Enzyme buffer components may be optimized as required. This is Enzyme Buffer H Part# 2349 (BellBrook Labs) and 1 mM DTT. |
| Assay Plates | Assay Plates Can Be Purchased Directly From BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
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