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Why Transcreener?
9Direct detection, far red fluors: less interference
9Universal: Any Kinase, Any substrate, Any ATP concentration
9Sensitive: low substrate consumption, use less enzyme
9Single addition, mix and read format: easy automation
9Three fluorescent readouts, instrument‐validated: flexibility, confidence
9> 12hr reagent and signal stability: easy automation
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2
Why Transcreener?
Most kinase assay methods rely on phosphopeptide
detection and are either 1) not homogenous; ie, they
require a separation step, or 2) not universal; ie, they
require different assay reagents for different
phosphorylated products.
Binding to Antibody
(FP, TR-FRET, EFC)
Binding to
Immobilized Metal
(FP, TR-FRET)
Acceptor
Kinase
ATP
Coupled
Enzyme
Assay
(Abs, Lum)
ADP
Coupled
Enzyme Assay
(Abs, FI)
Acceptor–OPO3
Radioassay
(SC, SP)
Reduced Protease
Sensitivity
Electrophoretic
(FRET)
Separation
(FI)
ADP or ATP detection methods are universal,
however they rely on coupling enzymes, which are
subject 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 of
ADP. Binding of tracer to antibody causes
a change in fluorescence. There are just
two components, and no intermediate
steps.
All other ADP assays are indirect and more
complex; ADP is converted to a detectable
product in a series or enzymatic steps,
each of which is subject to inhibition by
library compounds.
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Three far red fluorescent readouts provide plate
reader options and flexibility.
The Transcreener ADP Assay is available in FP, TR-FRET and FI format. For all three
assays, displacement of tracer from Ab by ADP causes a change in the fluorescence signal.
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True mix and read detection format,
endpoint or continuous detection.
The assays are true mix-and-read format,
with the enzyme quenching and ADP
detection components added as a single
reagent for endpoint assays. The assay
can also be used in a continuous detection
mode, which makes optimization of
enzyme reactions simpler.
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Comparison of ADP Detection Assays
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Validation in peer reviewed studies
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Transcreener™ ADP2 Assay:
Universal detection of kinases, acceptors
225
Peptide Substrates
200
Protein Substrates
cdk5/p35 (Histone 1)
COT (MEK1)
p38alpha (MBP)
PKA (histone H1)
PKA (MBP)
RAF1 (MEK1)
Δ mP
Abl1 (Abltide)
175
AKT/PKB (Akt/SKG peptide)
PKA (kemptide)
150
125
100
75
50
25
0
0
200
400
600
800
1000
Kinase Concentration (ng/mL)
Universal detection means you can use any kinase and any acceptor
substrate, including native proteins , which provide a more physiologically
relevant measure of kinase activity.
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Transcreener™ ADP2 Assay:
Universal detection of kinases, acceptors
200
175
150
Δ mP
125
Lipid Kinases
100
PI3 (phosphatidylinositol diP)
sphingosine 1 (D-sphingosine)
75
50
Metabolic Kinases
hexokinase (glucose)
phosphofructokinase (Fruc-6-P)
25
0
0
400
800
1200
1600
2000
Kinase Concentration (ng/mL)
Universal detection means straightforward detection
carbohydrate kinases in addition to protein kinases.
of
lipid
and
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Transcreener™ ADP2 Assay:
Universal detection of non-kinase ATP utilizing enzymes
200
175
150
EC80 = 4 ng/mL
ΔmP= 140 units
EC80 = 20 ng/mL
ΔmP= 160 units
Δ mP
125
100
75
Human+ATP
Human-ATP
Mouse+ATP
Mouse-ATP
50
25
0
0.01
0.1
1
10
100
1000
[P97], ng/mL
Universal detection also includes any of the thousands of
enzymes that use ATP to drive cellular reactions including
p97 ATPase (shown above) chaperonin ATPases, Acetyl CoA
carboxylase, and RecA.
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Transcreener ADP2 Assay Sensitivity:
Z′ > 0.7 at ≤ 10% ATP Conversion
1000μM
100μM
10μM
1μM
0.1μM
50000
40000
30000
1.5
1000μM
100μM
10μM
Δ Ratio 670/620
ΔmP
200
60000
RFU
300
TR-FRET Assay
FI Assay
FP Assay
1μM
0.1uM
20000
100
10000
0
0.0001 0.001 0.01
0.1
1
10
100 1000
0
0.0001 0.001 0.01
ADP μM
Transcreener FP
Transcreener TR‐FRET
Transcreener FI
Luc‐ADP Detection Assay
Luc‐ATP Depletion Assay
0.1
1
10
100 1000
ADP, μM
1 µM ATP/ADP standard curve
LLD (µM)
Z' at 10% Conv
0.86
0.02 ±0.07
0.71
0.10 ±0.06
0.92
0.03 ±0.01
ND
0.40 ±0.87
0.25 ±0.40
ND
10 µM ATP/ADP standard curve
Z' at 10% Conv
LLD(µM)
0.85
0.01 ±0.12
0.72
0.10 ±0.09
0.88
0.05 ±0.04
0.30
0.50 ±0.32
ND
1.50 ±0.30
1.2
0.9
0.6
1000μM
100μM
10μM
1μM
0.1μM
0.3
0.0
0.0001 0.001 0.01
0.1
1
10
100 1000
ADP μM
100 µM ATP/ADP standard curve
Z' at 10% Conv
LLD(µM)
0.89
1.0 ±0.3
0.72
1.0 ±0.3
0.92
0.5 ±0.4
0.62
5.0 ±0.7
0.52
7.0 ±0.6
Standard curves for conversion of ATP to ADP demonstrate robust detection at 10% conversion
starting at ATP concentrations from 0.1 to 1,000 μM. By comparison, Luciferase-based ADP and ATP
detection methods require greater conversion of ATP, especially at lower starting concentrations;
this translates into higher enzyme consumption.
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Transcreener ADP2 Assay Sensitivity Allows
Use of Submicromolar ATP
ZAP 70, 0.1 μ M AT P
8.0% ATP Conversion
Z' = 0.74
300
250
mP
200
150
100
11.7 ng/ml ZAP70
no enzyme control
50
0
0
5
10
15
20
25
Replicate Number
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Transcreener ADP2 FP Assay:
Overnight Reagent and Signal Stability
21 Day Reagent Stability
250
Control
-80°C
-20°C
4°C
RT
37°C
150
100
1hr
4hr
8hr
24hr
150
100
50
50
0
24 hr Signal Stability
200
Δ mP
200
ΔmP
250
0.01
0.1
1
ADP (µM)
10
0
0.01
0.1
1
10
ADP (µM)
Standard curves for conversion of 10uM ATP to ADP demonstrate the outstanding stability of
Transcreener detection reagents prior to addition to reaction and the stability of the signal
following addition to kinase reactions. Data is for the FP assay, the FI and TR-FRET assays
also have at least overnight reagent and signal stability. This provides outstanding flexibility
for automated HTS platforms, especially with large numbers of plates, where there may be a
lag between addition of detection reagent and plate-reading.
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9Direct ADP detection
9Universal for any kinase, ATPase, or substrate
9Three fluorescent detection formats
9Single addition, mix and read format
9Low nanomolar sensitivity
9Overnight stability
For more information on the Transcreener ADP2 Assays, email us at
[email protected] or call toll free 866-313-7881.
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Four assays, thousands of targets
GAPs
(CMP)
Methyltransferase
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