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Direct detection, far red fluors: less interference

Universal: Any Kinase, Any substrate, Any ATP concentration

Sensitive: low substrate consumption, use less enzyme

Single addition, mix and read format: easy automation

Three fluorescent readouts, instrument‐validated: flexibility, confidence

> 12hr reagent and signal stability: easy automation

Why Transcreener?

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Why Transcreener?

Acceptor–OPO3

ATP ADP

Binding to Antibody (FP, TR-FRET, EFC) Binding to

Immobilized Metal (FP, TR-FRET)

Reduced Protease Sensitivity

(FRET)Electrophoretic

Separation (FI)

Coupled Enzyme Assay

(Abs, Lum)

Coupled Enzyme Assay

(Abs, FI)

Radioassay(SC, SP)Acceptor Kinase

Most kinase assay methods rely on phosphopeptidedetection and are either 1) not homogenous; ie, theyrequire a separation step, or 2) not universal; ie, theyrequire different assay reagents for differentphosphorylated products.

ADP or ATP detection methods are universal,however they rely on coupling enzymes, which aresubject to interference from library compounds.

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Transcreener ADP2 Assay: Direct detection of ADP means less chance for interference

Transcreener relies on direct detection ofADP. Binding of tracer to antibody causesa change in fluorescence. There are justtwo components, and no intermediatesteps. All other ADP assays are indirect and more

complex; ADP is converted to a detectableproduct in a series or enzymatic steps,each of which is subject to inhibition bylibrary compounds.

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The Transcreener ADP Assay is available in FP, TR-FRET and FI format. For all threeassays, displacement of tracer from Ab by ADP causes a change in the fluorescence signal.

Three far red fluorescent readouts provide plate reader options and flexibility.

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The assays are true mix-and-read format,with the enzyme quenching and ADPdetection components added as a singlereagent for endpoint assays. The assaycan also be used in a continuous detectionmode, which makes optimization ofenzyme reactions simpler.

True mix and read detection format, endpoint or continuous detection.

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Comparison of ADP Detection Assays

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Validation in peer reviewed studies

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0 200 400 600 800 10000

25

50

75

100

125

150

175

200

225

Abl1 (Abltide)AKT/PKB (Akt/SKG peptide)PKA (kemptide)

PKA (MBP)

p38alpha (MBP)

cdk5/p35 (Histone 1)COT (MEK1)

RAF1 (MEK1)

Protein Substrates

Peptide Substrates

PKA (histone H1)

Kinase Concentration (ng/mL)

Δ m

P

Transcreener™ ADP2 Assay: Universal detection of kinases, acceptors

Universal detection means you can use any kinase and any acceptorsubstrate, including native proteins , which provide a more physiologicallyrelevant measure of kinase activity.

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0 400 800 1200 1600 20000

25

50

75

100

125

150

175

200

PI3 (phosphatidylinositol diP)

hexokinase (glucose)phosphofructokinase (Fruc-6-P)

sphingosine 1 (D-sphingosine)

Lipid Kinases

Metabolic Kinases

Kinase Concentration (ng/mL)

Δ m

P

Transcreener™ ADP2 Assay: Universal detection of kinases, acceptors

Universal detection means straightforward detection of lipid andcarbohydrate kinases in addition to protein kinases.

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0.01 0.1 1 10 100 1000

0

25

50

75

100

125

150

175

200

Human+ATPHuman-ATPMouse+ATPMouse-ATP

EC80 = 20 ng/mLΔmP= 160 units

EC80 = 4 ng/mLΔmP= 140 units

[P97], ng/mL

Δm

P

Transcreener™ ADP2 Assay: Universal detection of non-kinase ATP utilizing enzymes

Universal detection also includes any of the thousands ofenzymes that use ATP to drive cellular reactions includingp97 ATPase (shown above) chaperonin ATPases, Acetyl CoAcarboxylase, and RecA.

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Transcreener ADP2 Assay Sensitivity: Z′ > 0.7 at ≤ 10% ATP Conversion

0.0001 0.001 0.01 0.1 1 10 100 10000

100

200

3001000μM100μM10μM1μM0.1μM

ADP μM

Δm

P

0.0001 0.001 0.01 0.1 1 10 100 10000.0

0.3

0.6

0.9

1.2

1.51000μM100μM10μM1μM0.1μM

ADP μM

Δ R

atio

670

/620

0.0001 0.001 0.01 0.1 1 10 100 10000

10000

20000

30000

40000

50000

600001000μM100μM10μM1μM0.1uM

ADP, μMR

FU

Z' at 10% Conv LLD (µM) Z' at 10% Conv LLD(µM) Z' at 10% Conv LLD(µM)Transcreener FP 0.86 0.02 ±0.07 0.85 0.01 ±0.12 0.89 1.0  ±0.3

Transcreener TR‐FRET 0.71 0.10 ±0.06 0.72 0.10 ±0.09 0.72 1.0 ±0.3Transcreener FI 0.92 0.03 ±0.01 0.88 0.05 ±0.04 0.92 0.5 ±0.4

Luc‐ADP Detection Assay ND 0.40 ±0.87 0.30 0.50 ±0.32 0.62 5.0 ±0.7Luc‐ATP Depletion Assay ND 0.25 ±0.40 ND 1.50 ±0.30 0.52 7.0 ±0.6

1 µM ATP/ADP standard curve 10 µM ATP/ADP standard curve 100 µM ATP/ADP standard curve

Standard curves for conversion of ATP to ADP demonstrate robust detection at 10% conversionstarting at ATP concentrations from 0.1 to 1,000 μM. By comparison, Luciferase-based ADP and ATPdetection methods require greater conversion of ATP, especially at lower starting concentrations;this translates into higher enzyme consumption.

FP Assay FI Assay TR-FRET Assay

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ZAP 70, 0.1 μM ATP8.0% ATP Conversion

Z' = 0.74

0 5 10 15 20 250

50

100

150

200

250

300

11.7 ng/ml ZAP70no enzyme control

Replicate Numberm

P

Transcreener ADP2 Assay Sensitivity Allows Use of Submicromolar ATP

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0.01 0.1 1 100

50

100

150

200

250

1hr4hr8hr24hr

ADP (µM)Δ

mP

0.01 0.1 1 100

50

100

150

200

250

RT37°C

Control-80°C-20°C4°C

ADP (µM)

Δm

PTranscreener ADP2 FP Assay: Overnight Reagent and Signal Stability

24 hr Signal Stability21 Day Reagent Stability

Standard curves for conversion of 10uM ATP to ADP demonstrate the outstanding stability ofTranscreener detection reagents prior to addition to reaction and the stability of the signalfollowing addition to kinase reactions. Data is for the FP assay, the FI and TR-FRET assaysalso have at least overnight reagent and signal stability. This provides outstanding flexibilityfor automated HTS platforms, especially with large numbers of plates, where there may be alag between addition of detection reagent and plate-reading.

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For more information on the Transcreener ADP2 Assays, email us at info@bellbrooklabs.com or call toll free 866-313-7881.

Direct ADP detectionUniversal for any kinase, ATPase, or substrateThree fluorescent detection formatsSingle addition, mix and read formatLow nanomolar sensitivityOvernight stability

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Four assays, thousands of targets

Methyltransferase

GAPs

(CMP)