JAK3 Assay – Transcreener Application

Learn More About Transcreener Method Used for This JAK3 Assay

JAK3 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Kinase Assay directly measures ADP produced by JAK3 (Janus Kinase 3). These ADP measurements allow researchers to effectively determine the activity of the JAK3 enzyme. The assay provides a powerful tool to screen compound libraries for JAK3 modulators to help find new therapies for disease. 

The kit comes complete with all the detection reagents required to measure activity. JAK3 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

How Does This JAK3 Assay Work?

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.

Direct Detection of ADP to Measure JAK3 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener JAK3 Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

ADP FI Kinase Assay Kit

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

ADP TR-FRET Kinase Assay Kit

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of JAK3
  • Screen Compound Libraries for JAK3 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The JAK3 assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

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.

Detection of ADP Under JAK3 Initial Velocity - FP Readout

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.

Human JAK3 Enzyme Titration

JAK3 Assay Enzyme Titration

Linear Response

Screen for Inhibitors & Perform SAR

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.

Dose-Response - Staurosporine

JAK3 Staurosporine Inhibitor Titration

IC50 = 1.814 nM

Three Fluorescent Readouts Available

Choose the readout that is the best fit for your lab based on preference and plate reader compatibility. 

Fluorescent Intensity

JAK3 Enzyme Titration FI Readout

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.

TR-FRET

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.

JAK3 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your JAK3 program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewIn Review
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

Transcreener ADP2 Assay Kits - For JAK3 Activity Assays

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Cat. No.Product DescriptionPriceAdd to Cart
3013-ATranscreener ADP2 FI Assay (200 Assay, 96 Well)$359.00Add to Cart
3013-1KTranscreener ADP2 FI Assay (1,000 Assay, 384 Well)$759.00Add to Cart
3013-10KTranscreener ADP2 FI Assay (10,000 Assay, 384 Well)$4,299.00Add to Cart
3013-100KTranscreener ADP2 FI Assay (10 X 10,000 Assay, 384 Well)$21,495.00Add to Cart
3010-1KTranscreener ADP2 FP Assay (1,000 Assay, 384 Well)$725.00Add to Cart
3010-10KTranscreener ADP2 FP Assay (10,000 Assay, 384 Well)$4,099.00Add to Cart
3010-100KTranscreener ADP2 FP Assay (10 X 10,000 Assay, 384 Well)$20,495.00Add to Cart
3011-1KTranscreener ADP2 TR-FRET Red Assay (1,000 Assay, 384 Well)$789.00Add to Cart
3011-10KTranscreener ADP2 TR-FRET Red Assay (10,000 Assay, 384 Well)$4,449.00Add to Cart
3011-100KTranscreener ADP2 TR-FRET Red Assay (10 X 10,000 Assay, 384 Well)$22,245.00Add to Cart

Assay Plates for FI & FP

Cat. No.Product DescriptionPriceAdd to Cart
2233Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)$55.00Add to Cart
2229Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)$60.00Add to Cart
2230Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)$479.00Add to Cart

Assay Plates for TR-FRET

Cat. No.Product DescriptionPriceAdd to Cart
2234Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)$55.00Add to Cart
2231Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)$65.00Add to Cart
2232Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)$529.00Add to Cart

The Role of JAK3 As Therapeutic Target

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.

Related Resources

Related Resources
Transcreener ADP Kinase Assay Reviews

BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400

info@bellbrooklabs.com

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