The Transcreener JAK1 Assay determines JAK1 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 JAK1 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 a fluorescent readout.
The JAK1 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 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized JAK1 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. Both reactions have 10 µM ATP, 10 µM IRS1 peptide substrate for an enzyme reaction of 60 minutes at 30°C. Z’ shown here = 0.79.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 10 µM ATP with 10 µM IRS1(Y608) peptide substrates. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The JAK1 enzyme reaction took place for 60 minutes at 30°C. 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 8 nM JAK1, 10 µM ATP, 10 µM IRS1 peptide substrate for an enzyme reaction of 60 minutes at 30°C.
Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.
Assay conditions for the JAk1 titration shown include 10 µM ATP, 10 µM IRS1 Peptide, and 11.8 µg/mL ADP² antibody in the detection mixture.
Assay conditions for the JAK1 titration shown include 10 µM ATP, 10 µM IRS1 peptide, 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 JAK1. |
| Component | Notes |
|---|---|
| JAK1 Enzyme | We have successfully used recombinant human JAK1 from SignalChem Cat. #J01-11G and used JAK1 at 8 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 IRS1(Y608) Peptide at 10 µM (SignalChem Cat. # I40-58). |
| 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. |
Signal transducer and activator of transcription (STAT) proteins are transcription factors that lead to cellular immunity, proliferation, apoptosis, and differentiation though chain reactions. Once a cytokine binds to its receptor, JAK proteins activate and phosphorylate the receptor. This allows subsequent STAT binding and relocation to the nucleus where transcription occurs.
In normal cells, this pathway enhances the immune response to fight diseases. Genome studies found a multitude of polymorphisms in many of the proteins involved in this pathway which contribute to autoimmune and inflammatory disorders. One current JAK1 inhibitor Upadacitinib (Rinvoq) is currently marketed for Rheumatoid Arthritis treatment by AbbVie.
The Transcreener ADP² JAK1 Assay is an excellent tool for researchers examining the therapeutic effects of JAK1.
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