Based on HTRF technology, the HTRF Transcreener ADP assay is a universal method for identifying and characterizing the phosphotransferase activity induced by any ATP/ADP dependent target, such as serine/threonine and tyrosine kinases, as well as lipid kinases and chaperonins.
The formation of ADP is detected by a specific monoclonal antibody labeled with Eu3+ cryptate, and directly correlates with the amount of phosphorylated substrate in kinase assays.
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.
For kinases, the substrate is first incubated with the kinase. Then the addition of ATP allows the enzymatic reaction to start: the kinase phosphorylates the substrate.
For ATPases, ATP is converted into ADP and inorganic phosphate.
The anti-ADP labeled with Eu3+ cryptate competes with native ADP and d2-labeled ADP. As a result, the signal is inversely proportional to the concentration of ADP in the sample.
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.
Figure 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 concentrations of ADP or phosphorylated substrate were calculated using the appropriate standard curves.
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 been run successfully. The results shown in figure 2 demonstrate the ability to use a broad range of ATP concentrations while maintaining assay performance.
Figure 2: ATP/ADP standard curves were generated from 1μM to 500 μM. To mimic ADP generation during an enzymatic reaction, the total adenosine concentration remained constant for each sample within the standard curves, while the percentage of ADP within each sample varied.
As shown here, 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.
Figure 3 : The IC50 was found at 0.87μM. This value is in the same range as that published in Salvatore DeBonis’ paper* (1μM).
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| HTRF Transcreener ADP kit - 1,000 tests | 62ADPPEB | ||
| HTRF Transcreener ADP kit - 20,000 tests | 62ADPPEC |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| HTRF Transcreener ADP kit d2 conj - 1,000 tests | 62ADPXDB | - | - |
| HTRF Transcreener ADP kit d2 conj - 20,000 tests | 62ADPXDC | - | - |
| HTRF Transcreener ADP detection buffer - 13 ml | 62DB1RDD | - | - |
| HTRF Transcreener ADP detection buffer - 200 ml | 62DB1RDF | - | - |
| HTRF Transcreener ADP Enzymatic buf. 5X - 10 mL | 62ZB1FDC | - | |
| HTRF Transcreener ADP Enzymatic buf. 5X - 50 mL | 62ZB1FDD | - | - |
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