| Assay Method Information | |
| | ENPP1 Assay |
| Description: | Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) hydrolyzes ATP, breaking it down into 5′-AMP and pyrophosphate. The 5′-AMP formed from the reaction is detected by Transcreener® AMP2/GMP2 TR-FRET Assay (Bellbrook), containing AMP-tracer (HiLyte 647) and anti-AMP Terbium (Tb) donor. 5′-AMP generated in the reaction competes with AMP-tracer for binding with Anti-AMP Tb donor, and decreases TR-FRET signal. Different concentrations of ENPP1 inhibitors are added to dry assay plate, followed by 2 μl 300 nM ATP. The reaction is initiated with the addition of 2 μl 8.5 μM human ENPP1 (generated by SBP Protein group). The assay reaction mixture contains a buffer of 50 mM Tris, pH 7.5, 1 mM TCEP, 2 mM MgCl2, 0.005% Tween 20, and 1% DMSO. The reaction is stopped after 1 hour at room temperature by adding 2 μL of 2 nM AMP-tracer and 2 μl of 2 nM Anti-AMP Tb donor in the 50 mM Tris, pH 7.5, buffer containing 20 mM EDTA and 0.1% Prionex. After 2 hour additional incubation at room temperature, the generated signal is measured using BMG Lab PHERASTAR instrument. |
| Affinity data for this assay | |
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