The Transcreener ADP² Assay uses a highly selective ADP antibody and far-red tracer to directly detect ADP produced by SRC. 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 SRC kinase assay kit comes complete with the detection reagents required to measure activity. SRC enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.
The enzyme reaction begins with the addition & incubation of SRC enzyme and substrate. Transcreener ADP Antibody & Tracer are then added & incubated. A compatible plate reader reads the plate.
Z’ data confirms the robustness of the SRC kinase assay. A robust assay is necessary to ensure accuracy and efficiency in order to scale up for high-throughput screening. The Z’ shown here demonstrates the assay is optimal for HTS.
Components in the 10 µL reaction included 25 mM HEPES (pH 7.5), 0.01% Brij, 1 mM EGTA, 10 mM MgCl2, 0.25 mg/mL Poly (4:1 Glu Tyr) substrate, and 160 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 87.4 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 SRC kinase 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, & 75.1 μg/mL ADP Ab-IRDye QC-1
TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 159.8 nM ADP HiLyte647 tracer.
Interested in moving your kinase 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 - 87.4 μg/mL, TR-FRET - 8 nM, and FI - 75.1 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 - 159.8 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 |
|---|---|
| SRC-Kinase Enzyme | We have successfully used Human SRC, His Tag Recombinant Protein from ThermoFisher Scientific Cat. #P3044. |
| Poly (4:1 Glu, Tyr) Peptide | We have successfully used Poly (4:1 Glu, Tyr) Peptide from SignalChem Biotech Cat. #P61-58. |
| Enzyme Buffer | 25 mM HEPES (pH 7.5), 0.01% Brij, 1 mM EGTA, 10 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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BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
