The NDP (nucleoside diphosphate) Sensor is a fluorescent sensor protein for the development of universal kinase assays, compound profiling, and substrate identification.
A Universal Tool for Kinase Assays
- Universal kinase compatibility—wide range of nucleotide concentrations and protein, peptide, or lipid substrates
- Fast and accurate—rapidly responds to ATP/ADP ratio
- Enables endpoint or real-time kinase assays
The NDP Sensor is a coumarin-labeled form of a bacterial NDP kinase. The sensor autophosphorylates a histidine residue in the presence of NTP (nucleoside triphosphate), producing a highly fluorescent P~NDP sensor (Figure 1). The reverse reaction regenerates the unphosphorylated form. In the presence of excess nucleotides over the NDP Sensor, the ATP/ADP ratio determines the NDP Sensor phosphorylation state via fluorescence intensity.
Table 1. Z´-LYTE™-compatible monochromator-based instruments
| || Tecan Safire and Safire2™ |
|Excitation filter ||400/12* |
|Emission filter, donor ||445/12* |
|Emission filter, acceptor ||520/12*|
*Wavelength in nm/bandpass
Mechanism of the NDP Sensor
The NDP Sensor is an excellent universal kinase assay platform for compound profiling and screening (Figure 2), either in endpoint or kinetic mode, and can also be used to identify substrates for uncharacterized kinases (Figure 3).
Table 2. Z´-LYTE™-compatible filter-based instruments
| Tecan GENios Pro™ || Tecan Ultra™ series || Molecular Devices Analyst® || PerkinElmer EnVision™ |
|Excitation filter ||405/20* ||405/20* ||405/35* ||400/25* |
|Emission filter, donor ||465/35* ||465/35* ||460/40* ||460/25* |
|Emission filter, acceptor ||535/20* ||535/20* ||530/25* ||535/25* |
|Dichroic for excitation ||330/420:440/850 ||50% ||425 ||General dual |
|Dichroic for emission ||330/420:440/850 ||320/500:520/800 ||425 ||General dual|
*Wavelength in nm/bandpass
Figure 2. The NDP Sensor as a universal kinase assay
The NDP Sensor enables endpoint or real-time kinase assays with peptide, protein, or lipid substrates. All fluorescence measurements were taken at 440/487 nm (Ex/Em) in 384-well plates.
A. For PKA, 20 μl reactions were monitored continuously with 10 μM ATP, 50 μM Crosstide, and 0.25 μM NDP Sensor.
B. and C. For endpoint reads with p38α, 60 min kinase reactions were performed with 10 μM ATP and either 0.75 mg/ml myelin basic protein (MBP), 50 μM EGF-R peptide, or 500 μM EGF-R peptide, in 10 μl. For endpoint reads, 10 μl of 0.5 μM NDP Sensor was added to the reactions prior to measurements.
D. Endpoint reactions for PI3 Kinase were performed as for p38α, except that the reactions were incubated for 100 min, with 50 μM L-α-phosphatidylinositol-4,5-bisphosphate as the substrate.
Using the NDP Sensor for kinase substrate identification