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.

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

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

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

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

Assay Plates for TR-FRET

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.

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