The Transcreener JAK3 Assay determines JAK3 enzyme 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 JAK3 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 JAK3 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.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The JAK3 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized JAK3 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 = 0.75.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 10 µM ATP with 20 µM JAK3tide peptide substrates. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The JAK3 enzyme reaction took place for 60 minutes at room temperature. The enzymatic reaction was then halted using Stop & Detect Buffer B included in the kit and assay plates were read. Non-productive hydrolysis of ATP occurs in many kinase reactions due to many factors including autophosphorylation and water exploiting the ATP binding pocket of the enzyme.
Transcreener Assays are designed for screening 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 include 3 nM JAK3, 10 µM ATP, 0.2 µg/µL Poly(Glu-Tyr, 4:1) substrate for an enzyme reaction of 60 minutes at room temperature.
Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.
Assay conditions for the JAk3 titration shown include 10 µM ATP, 0.2 µg/mL Poly (Glu-Tyr, 4:1), and 11 µg/mL ADP² antibody in the detection mixture.
Assay conditions for the JAK3 titration shown include 10 µM ATP, 0.2 µg/mL Poly (Glu-Tyr, 4:1), 27 nM tracer, and 8 nM ADP² Tb Antibody in the detection mixture.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration (in the Detection Mixture) for FP is 11.8 μg/mL for 10 μM ATP. 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 B, 10X | 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. We suggest using a starting ATP concentration of 10 μM for JAK3. |
| Component | Notes |
|---|---|
| JAK3 Enzyme | We have successfully used recombinant human JAK3 from SignalChem Cat. #J03-11G and used JAK3 at 3 nM for screening purposes. |
| Enzyme Buffer | 20 mM Tris (pH 7.5), 5 mM MgCl2,, 0.01% Brij-35. Enzyme buffer components may be optimized as required. |
| Substrate | Use the substrate to be phosphorylated for your experiment. Here we used Poly (Glu-Tyr, 4:1) Peptide at 0.2 µg/µL (SignalChem Cat. # P61-58) or JAK3tide at 20 µM (SignalChem Cat. # J03-58) |
| 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. |
JAK3 is a tyrosine kinase that activates via cytokine receptors (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). JAK3 also autophosphorylates in the presence of ATP; examples of that can be seen in the data below. JAK3 phosphorylates and activates STATs that induce the expression of genes in the cell nucleus.
Recent research has shown that JAK3 activation is common in many different cancers and that JAK3 autophosphorylation could be a rate-limiting step. JAK3 mutations have been identified in different forms of leukemia and lymphoma. Inhibiting JAK3 or JAK1 could provide a potential treatment for these cancers.
Because it is expressed only in hematopoietic cells (which should help to limit off-target effects), JAK3 is also being vetted for autoimmune diseases like rheumatoid arthritis. Tofacitinib, a JAK1 and JAK3 inhibitor, is currently marketed by Pfizer. New specific JAK3 inhibitors could help limit the current off-target effects of the drug.
The Transcreener ADP2 JAK3 Assay is an excellent tool for researchers examining the therapeutic effects of JAK3.
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