Biology at Work
Subscribe to updatesTranscreener® ADP2 FP Assay
Still Universal. Now More Sensitive.
Introductory offer! – from now through November 30, 2008 your research dollars will go twice as far. When you mention code W1008 you can enjoy the Transcreener ADP2 FP Assay at 50% off (1,000 reaction kit only). Call now. 866.313.7881
| Product | Cat# | Size | Price |
|---|---|---|---|
| Transcreener ADP2 FP Assay | 3010-1K | 1000 assays | $585 |
| Bulk Ordering | |||
| For orders of 10,000 wells or more, please contact us for a price quote. info@bellbrooklabs.com | |||
Click logo for Instrument Specific FP Validation Information
- Transcreener ADP2 FP Assay Technical Manual
- Transcreener ADP Posters and Presentations
- Transcreener Technical Resources
Benefits:
- Increased Sensitivity
- Use Less Enzyme and Substrate
- ADP production at reported ATP K m
- ADP production at below reported ATP K m
- Excellent Z' Values at Initial Enzyme Velocity Kinetics
- Miniaturize to 1536
- Robust Performance
The Transcreener ADP2 FP Assay is a new assay, with greater sensitivity than the original Transcreener ADP Assay. The improvement is a more sensitive antibody against ADP yielding an excellent signal at less than or equal to 10% ATP consumption for a broad range of initial ATP concentrations (0.1-1,000 μM). The result is the ability to screen low ATP K m enzymes, and to use initial velocity enzyme kinetics at or below ATP K m concentrations, which leads to accurate inhibitor potencies and the ability to use less enzyme and substrate. Ratiometric, red-shifted fluorescence polarization output minimizes signal variability and reduces compound interference, resulting in robust Z´ values ≥ 0.7 in 384 and 1536-well formats.
Like its predecessor, the Transcreener ADP2 FP Assay is a homogenous fluorescence polarization assay that enables the facile detection and screening of established drug targets including protein and lipid kinases as wells as emerging targets such as heat shock proteins and other ATPases. The assay is based on the immunodetection of ADP and allows the screening of diverse enzymes with native and synthetic substrates, or enzymes with intrinsic ATPase (no substrate) – all with the same set of reagents. As an added benefit, using the PerkinElmer EnVision® microplate reader will no longer require modified assay conditions for outstanding results.
The Transcreener ADP2 FP Assay was developed to measure the progress of any enzyme that produces ADP. The tracer is displaced by ADP, the invariant product generated during the enzyme reaction, resulting in a decrease in fluorescence polarization. The assay uses a red-shifted tracer which reduces compound interference and provides a robust ratiometric readout.
- UNIVERSAL: Homogenous ADP detection
- FLEXIBLE: Any ADP-producing enzyme and substrate.
- RELEVANT: Rapid pharmacologically relevant data.
- SENSITIVE: Z' > 0.7 at low ATP conversion.
- SMART: One set of reagents reduces time and expense.
Increased Sensitivity-Broader Range of Detection
This assay (cat #3010) has greater sensitivity, resulting in a broader range of ADP detection than the original Transcreener ADP Assay (cat #3004)
Figure 1. A broader range of starting ATP concentrations is achieved with the improved ADP2 Antibody. To mimic ADP generated during an enzyme reaction, standard curves optimized for 0.1 µM, 1 µM, 10 µM, 100 µM, and 1,000 µM ATP. The standard curve mimics an enzyme reaction: as ATP concentration decreases, ADP concentration increases. The adenine concentration remains constant. Assay buffer conditions were optimized for each antibody/tracer pair. The EC85 antibody concentration for both antibodies was used for each initial ATP concentration (n=24).
Use Less Enzyme and Substrate- Screen Enzymes at or Below their ATP Km
Fig 2A) ADP Production at Reported ATP Km
Figure 2A. ADP production by protein kinases using the reported ATP Km concentration. Kinases were serially diluted in
4 mM MgCl2, 2 mM EGTA, 50 mM HEPES, 0.01% Brij-35, 1% DMSO, and 6 µM Poly Glu 4:1 substrate (ZAP70), 150 µM S6 kinase substrate (ROCK1), or 250 µM ABL cytosolic substrate (ABL1 T315I). Fluorescence polarization was read on an PerkinElmer EnVision® plate reader. Error bars represent the standard error of the mean (n=3),
ΔmP= mPno enzyme - mPenzyme.
Fig 2B) ADP Production at 0.1 µM ATP
Figure 2B. ADP production by ZAP70 and ABL1T315I using 0.1 µM ATP. Kinases were serially diluted in 4 mM MgCl2, 2 mM EGTA, 50 mM HEPES, 0.01% Brij-35, 1% DMSO, and 6 µM Poly Glu 4:1 substrate (ZAP70) or 250 µM ABL cytosolic substrate (ABL1 T315I). Fluorescence polarization was read on an PerkinElmer EnVision® plate reader. Error bars represent the standard error of the mean (n=3) ΔmP= mPno enzyme - mPenzyme.
Excellent Z' Values at Initial Enzyme Velocity Kinetics
Fig 3A) Z' determination for ZAP70 performed at 0.1 μM ATP ( below reported ATP Km)
Fig 3B) Z' determination for ZAP70 performed at 2 μM ATP (at reported ATP Km)
Figure 3. Z' determinations for kinase reactions performed under initial rate conditions at low ATP concentrations. Reactions (10 μL) with and without enzyme
were prepared using the enzyme and ATP concentrations indicated above. ZAP70 concentration = 12 ng/mL.
A) ATP concentration = 0.1 μM; Z'-
value = 0.74; and % ATP Conversion = 8%.
B) ATP concentration = 2 μM; Z'- value = 0.71; and % ATP Conversion = 4.6%.
Miniaturize to 1536
The Transcreener ADP2 FP Assay is also well suited for miniaturization, providing excellent results in a 1536 well assay format.
Fig 4A) Standard curve using ADP2 Antibody at 0.1 μM ATP generated in a 1536 well plate
Fig 4B) Standard curve using ADP2 Antibody at 10 μM ATP generated in a 1536 well plate
Fig 4C) Standard curve using ADP2 Antibody at 1,000 μM ATP generated in a 1536 well plate
Figure 4. Z' and Δ mP values observed in a standard curve mimicking conversion of ATP to ADP. Total reaction volume equals 10 μL.
A) ∆ mP Cutoff = 90 mP and Z' Cutoff = 0.5. The dotted black line is set at 15% conversion of 0.10 μM ATP.
B) ∆ mP Cutoff = 95 mP and Z' Cutoff = 0.7. The dotted black line is set at 10% conversion of 10 μM ATP.
C) ∆ mP Cutoff = 95 mP and Z' Cutoff = 0.7. The dotted black line is set at 10% conversion of 1,000 μM ATP.
Robust Performance on Industry- Standard Microplate Readers
The Transcreener ADP2 FP Assay, like its predecessor, displays robust performance across industry-standard microplate readers containing filter sets or monochromator settings for red-shifted polarization. However, unlike the Transcreener ADP Assay, the new assay performs very well with the PerkinElmer EnVision® instrument without modified assay conditions.
Fig 5A. Standard Curve using ADP2 Antibody at 0.1 μM ATP
Fig 5B. Standard Curve using ADP2 Antibody at 1 μM ATP
Fig 5C. Standard Curve using ADP2 Antibody at 10 μM ATP
Figure 5. Z' and Δ mP values observed in a standard curve mimicking conversion of ATP to ADP as measured on a PerkinElmer Envision® Instrument. Assays were performed in 384 well plates. Data was generated using EC85 antibody concentrations and an instrument setting of 100 flashes (2:17 read time). The black dotted line represents 10% conversion of ATP to ADP. A) 0.1 µM ATP. B) 1 µM ATP. C) 10µM ATP.
For more information on the Transcreener ADP2 FP Assay contact BellBrook Labs:
866.313.7881 in North America
info@bellbrook labs.com