HTRF® Transcreener® ADP
For Kinase and ATPase screening
The HTRF® Transcreener® ADP assay is based on our patented HTRF® technology for the direct detection of ADP using a monoclonal antibody labeled with Eu3+ cryptate. HTRF is an enzyme-free detection chemistry thus producing fewer false positives. This assay can be used for high throughput screening or profiling of any ATP/ADP dependent target such as serine/threonine and tyrosine kinases as well as lipid kinases and chaperonins.
- Monoclonal antibody specific to ADP
- Homogeneous non-radioactive screening alternative
- Enzyme-free detection technology
- Compatible with any substrate
- Kinases and ATPases
- High throughput Screening
- Profiling
- Mechanistic studies
Assay Principle:
The HTRF® Transcreener® ADP assay is a competitive immunoassay in which native ADP and d2-labeled ADP compete for binding to a monoclonal anti-ADP labeled with Eu3+ cryptate. The assay consists of two steps: an enzymatic step followed by a universal detection step.
Enzymatic step:
For kinases, the substrate is incubated with the kinase. Then, the addition of ATP allows the enzymatic reaction to start: the kinase phosphorylates the substrate.
For ATPases, ATP will be converted into ADP and inorganic phosphate.
Detection step:
The anti-ADP labeled with Eu3+ cryptate will compete with native ADP and d2-labeled ADP. As a result, the signal is inversely proportional to the concentration of ADP in the sample.
Correlation between HTRF assay and radioactive γ-ATP method
Table 1 compares the level of ADP detected using the HTRF Transcreener ADP assay to a radioactive plate-based assay which measures the incorporation of labeled γ-phosphate onto a substrate. The assays were performed for the serine/threonine kinase CDK2/c2. The correlation of the two methods demonstrates the viability of using the HTRF ADP detection as an alternative to conventional ATP assays.
Table 1: Reactions were started with the addition of 100μM ATP. The kinase reaction was stopped at various times from 5 to 45 minutes with the addition of EDTA. HTRF detection reagents were added and emissions at 620 and 665 nm measured or radioactive ATP detected on a liquid scintillation plate reader. The concentration of ADP or phosphorylated substrate was calculated using the appropriate standard curves.
ATP concentration flexibility
The HTRF Transcreener ADP assay protocol can be tailored to use a range of ATP concentrations in collaboration with your dedicated scientific consultant. Assays performed using as little as 1μM ATP up to 500μM have successfully been run. The results shown on figure 3 demonstrate the ability to use a broad range of ATP concentrations while maintaining assay performance.
Figure 3: ATP/ADP standard curves were generated from 1μM to 500 μM. To mimic ADP generation during an enzyme reaction, the total adenosine concentration remained constant for each sample within the standard curves, while the percentage of ADP within each sample varied.
IC50 determination with the ATPase Eg5
As shown along side, HTRF Transcreener ADP assay was run with Eg5 ATPase and S-Trityl-L-Cysteine (Eg5 reference inhibitor) IC50 was calculated. Eg5 enzyme at 60 nM was first pre-incubated at 37°C for 1h30 in presence of various concentrations of S-Trityl-L-Cysteine from 500 μM to 30 nM, with a three fold dilution series between each concentration. Then ATP (100μM) and HTRF detection reagents were added and the plate incubated at room temperature for 30 minutes.
The IC50 was found at 0,87μM. This value is in the same range as that published in Salvatore DeBonis’ paper* (1μM).
Ordering Info:
| Description | Quantity* | Cat no. | Product Insert |
| HTRF® Transcreener® ADP | 1,000 tests 20,000 tests |
62ADPPEB 64ADPPEC |
pdf |
Companion Products:
| Description | Quantity* | Cat no. |
| HTRF® Transcreener® ADP enzymatic buffer 5x | 10mL | 62ZB1FDC |
| HTRF® Transcreener® ADP enzymatic buffer 5x | 50mL | 62ZB1FDD |
| HTRF® Transcreener® ADP detection buffer | 13mL | 62DB1RDD |
| HTRF® Transcreener® ADP detection buffer | 200mL | 62DB1RDF |
Transcreener® HTS Assay Platform technology is patented (U.S. patent 7,332,278), and is being used under license from Bellbrook Labs, LLC.
For more information or To Place an Order:
Europe and other countries
+33(0)466 796 705
