The Transcreener RIPK2 Assay determines RIPK2 activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.
Transcreener RIPK2 Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The RIPK2 assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
This application note describes methods for using the Transcreener ADP2 Kinase Assay to measure RIPK2 enzymatic activity in a high throughput format for inhibitor screening and/or inhibitor dose-response measurements.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The RIPK2 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized RIPK2 reaction conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z’ shown here = 0.74.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 50 μM ATP, 40 mM Tris (pH 7.5), 2.5 mM MnCl2, and 20 mM MgCl2. The enzyme buffer includes 40 mM Tris, 0.05 mM DTT, and 0.1 mg/mL BSA. The Detection Mix included 1X Stop & Detect Buffer B, 40 μg/mL ADP Antibody, and 4 nm Alexa 633 tracer. The RIPK2 enzyme reaction took place for 60 minutes at room temperature. The Detection Mix was added and incubated for 40 minutes at room temperature and read with a CLARIOstar plate reader.
Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.
FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 40 μg/mL ADP Ab-IRDye QC1, 8 nM AlexaFluor 594 Tracer.
TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 1000 nM ADP HiLyte647 tracer.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration for 10 μM ATP (in the Detection Mixture) with FP readout- 40 μg/mL, TR-FRET readout- 8 nM, and FI readout- 40 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. |
| 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. We suggest using a starting ATP concentration of 50 μM for RIPK2. |
| Component | Notes |
|---|---|
| RIPK2 Enzyme | We have successfully used recombinant human RIPK2 from SignalChem #R08-11G. A RIPK2 concentration of 0.12 μM was used for screening and SAR. |
| Enzyme Buffer | 40 mM Tris (pH 7.5), 0.1 mM DTT, 0.2 mg/mL BSA. Enzyme Buffer components may be optimized as required. |
| 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. |
RIPK2 plays an important role in regulating inflammatory signaling and cell-death pathways. NOD1 and NOD2 are pattern-recognition receptors that signal to RIPK2. Upon its recruitment by NOD1/2, RIPK2 activates NF-κB and MAPKs, producing pro-inflammatory cytokines (He & Wang 2018).
Overactivation of NOD1/2 can lead to autoinflammatory diseases, and RIPK2 inhibitors were found to significantly inhibit the immune response of these proteins. Recent studies also show involvement of RIPK2 in the occurrence and progression of several cancers, such as gastric cancer, breast cancer, and renal cell carcinoma (Song et al, 2022).
The Transcreener ADP RIPK2 Assay is an excellent tool for researchers examining the therapeutic effects of RIPK2.
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