Learn More About Transcreener Method Used for This JAK1 Assay

JAK1 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by JAK1 (Janus Kinase 1). These ADP measurements allow researchers to effectively determine the activity of the JAK1 enzyme. The assay provides a powerful tool to screen compound libraries for JAK1 modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. JAK1 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

How Does This JAK1 Assay Work?

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.

Direct Detection of ADP to Measure JAK1 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener JAK1 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-FET)

ADP TR-FRET Kinase Assay

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

Applications

  • Measure Enzymatic Activity of JAK1
  • Screen Compound Libraries for JAK1 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 assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

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.

JAK1 Assay Z'

Detection of ADP Under JAK1 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 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.

Human JAK1 Enzyme Titration

JAK1 Assay Enzyme Titration

Linear Response

JAK1 Assay Linear Enzyme Titration

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 8 nM JAK1, 10 µM ATP, 10 µM IRS1 peptide substrate for an enzyme reaction of 60 minutes at 30°C.

Dose-Response - Staurosporine

JAK1 Assay Dose Response Curve

IC50 = 3.417 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

JAK1 Assay Enzyme Titration FI

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.

TR-FRET

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.

JAK1 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 JAK1 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 JAK1 As a Therapeutic Target

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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