The Transcreener ADP² Assay uses a highly selective ADP antibody and far-red tracer to directly detect ADP produced by SMARCA2 (Aliases: BRM, BAF190, SNF2L2, NCBRS). Direct ADP detection simplifies the protocol to two steps (no coupling enzyme added) and reduces the risk of false positives and/or compound interference. The highly selective antibody allows ADP detection in the presence of excess ATP, a requirement for measuring enzyme initial velocity.
Transcreener Assays are designed for screening compound libraries in a high-throughput format, providing robust results ready to scale. Since their introduction in 2006, they have been used worldwide in millions of wells for inhibitor screening, profiling, and mechanistic studies in 96, 384, or 1536-well plates with most of the major multimode plate readers.
The SMARCA2 assay kit comes complete with the detection reagents required to measure activity. Enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.
Download the free poster, Interrogating SMARCA2 and SMARCA4 with the Transcreener ADP² Assay.
The Transcreener ADP² Assay relies on a simple, but robust mix-and-read procedure. Following an enzyme reaction, detection reagents containing ADP² antibody and tracer are added along with EDTA to quench the reaction. The plate is read with a compatible plate reader.
Z’ data confirms the robustness of the SMARCA2 assay. A robust assay is necessary to ensure accuracy and efficiency in order to scale up for high-throughput screening. The Z’ shown here (Z’ = 0.93) demonstrates the assay is optimal for HTS.
Components in the 10 µL reaction included 20 mM HEPES (pH 7.5), 0.005% Triton, 50 mM KCl, 1 mM DTT, 5 mM MgCl2, 0.52 mg/mL Salmon Sperm DNA, and 88 µM ATP. The enzyme reaction was incubated for 60 minutes at room temperature. The detection mix included 1X Stop & Detect Buffer B, 4 nM ADP tracer, and 96.04 µg/mL ADP2 antibody. The detection mix was incubated for 60 minutes at room temperature before reading the plate. These assay conditions are chosen to generate the best assay window and ability to characterize the enzyme. Raw polarization signal (mP) is converted to ADP formed using a standard curve.
The Transcreener HTS Assays are designed for screening compound libraries in a high throughput format and performing follow-up SAR to determine inhibitor potency.
Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. The SMARCA2 assay is available with an FP, FI, or TR-FRET readout. Reference our Instrument Compatibility resource to determine the detection mode compatible with your plate reader.
FI Detection Mix (10 μL): 1X Stop & Detect Buffer B, 8 nM AlexaFluor 594 Tracer, & 82.54 μg/mL ADP2 ADP Ab-IRDye QC-1
TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 175 nM ADP HiLyte647 tracer.
Interested in moving your ATPase program forward, but don’t want the hassle of bringing an assay in-house? BellBrook’s expert scientists have you covered. Our team of biochemical and enzymology expert scientists will work diligently to progress your lead candidates through the early-stages of drug discovery. The best part – we are a small custom shop. We can customize assay conditions to suit your project needs, produce results faster, and you will directly collaborate with one of our expert scientists.
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| Component | Notes |
|---|---|
| ADP2 Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration for 5 μM ATP (in the Detection Mixture) for FP - 96.04 μg/mL, TR-FRET - 8 nM, and FI - 82.54 μg/mL . Yours may vary depending on enzyme and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. The suggested concentration for 5 μM ATP (in the Detection Mixture) for FP - 4 nM, TR-FRET - 175 nM, and FI - 8 nM. |
| Stop & Detect Buffer | The Stop & Detect Buffer components will stop enzyme reactions that require Mg2+. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. |
| 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 |
|---|---|
| SMARCA2 Enzyme | We have successfully used Recombinant SMARCA2 (636-1131) protein from Active Motif Cat. #81439. We have also successfully used Full-Length SMARCA2 Chromatin Remodeling Enzyme (Human BRM) from EpiCypher Cat. #15-1015 |
| Salmon Sperm DNA | We have successfully used Invitrogen™ Ultrapure™ Salmon Sperm DNA Solution from Fisher Scientific Cat. #15-632-011. |
| Enzyme Buffer | Enzyme buffer components in the 10 µL reaction include 20 mM HEPES (pH 7.5), 0.005% Triton, 50 mM KCl, 1 mM DTT, and 5 mM MgCl2 |
| Assay Plates | Assay Plates Can Be Purchased Directly Through 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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