Omnia™ Kinase Principle—easier than 1–2–3.
|Omnia® Kinase Assays use the chelation-enhanced fluorophore (CHEF) called Sox, an unnatural amino acid, which is incorporated into a kinase-specific peptide substrate. Upon phosphorylation of the peptide by the kinase of interest, Mg2+ is chelated to form a bridge between the Sox moiety and the phosphate group that is added by the kinase to the specific tyrosine, serine or threonine residue on the peptide (Figure 1). Phosphorylation results in an increase in fluorescence (Figure 2). The fluorescence intensity is directly proportional to the amount of peptide phosphorylation. The protocol is easy to follow and delivers real-time data (Figure 3). Omnia® Kinase Assay is ideal for kinase inhibitor characterization (Figure 4). The assay offers high sensitivity (Figure 5) and high precision (Table 1) with minimal interference (Figure 6).|
Figure 1—Omnia™ Kinase Principle Upon phosphorylation, Mg2+ is chelated to form a bridge between the sox moiety and the phosphate group. Excitation at 360nm results in an increase in fluorescence emission at 485 nm.
|Figure 2—Emission spectra of Omnia® substrates. These emission spectra illustrate the 2-10 fold increase in fluorescence signal between the unphosphorylated substrate peptide (lower curve) and the fully phosphorylated peptide product (upper curve).|
Figure 3—Procedure for Recombinant and Lysate Assays. Simply mix the reaction, pipette into a 96-well plate, and read the fluorescence. Data can be collected every 30 seconds out to 1 hour, for instance.
|Figure 4—IC50 determination in crude cell lysates. Isoproterenol stimulated LNCaP cell lysates were assayed for PKA activity in the presence of increasing concentrations of PKI-tide. The estimated IC50 from this plot is 1 nM.|
|Figure 5—High sensitivity. The Omnia® Ser/Thr substrate 8 was used to measure the activity of recombinant PKCα at decreasing protein concentrations. 20% conversion of substrate to product was achieved at about 200 pM PKCα concentration.|
|Figure 6—Minimal interference. Interference from increasing concentrations of the auto fluorescent AKT inhibitor VIII (from 0 to 30,000 µM) is overcome by monitoring the rate of the reaction (change in fluorescent intensity over time) rather than an endpoint fluorescent signal. The IC50 value for this inhibitor was determined to be 94 nM.|
Table 1 - High precision
Z'Values: CV%, and Signal to Background/Noise Ratios for the Omnia® Assay for Akt at 10 µM substrate concentration.
|% Conversion||Signal to |
|Signal to |