ADP ACCUMULATION ASSAYS

Activity-Based ADP Accumulation Assays

ADP Hunter Plus and ADP Quest assays are homogeneous antibody-free assays for measuring ADP accumulation that are ideal for kinase profiling and high throughput screening applications. The ADP Hunter Plus assay is optimized for high throughput screening, compatible with reducing agents such as DTT, and applicable for endpoint detection of ADP. In contrast, the ADP Quest Assay is an ideal assay for kinase and inhibitor characterization to provide a fast, accurate tool for determining K_m, K_i, and mode-of-action, and applicable for kinetic or endpoint mode.

The assay uses enzymatic reactions that produce a signal that is directly proportional to the amount of ADP in the solution. These reactions utilize ADP to convert ADHP (10-Acetyl-3,7-dihydroxyphenoxazine), a fluorescent dye precursor, to fluorescent dye resorufin.

Gain Insight into Enzymology – Determine Kinase ATP K_m

Determination of Kinase ATP K_m. Michaelis-Menten plot for determining the ATP K_m (Michaelis-Menton constant) for protein kinase A (PKA) which provides insight into enzymology and mode of inhibition. This assay has K_m = 23 µM and R² = 0.990.

Characterize Kinase Activity in Both Kinetic and Endpoint Modes

Kinetic and endpoint modes kinase activity characterization. 
– (A.) PKA activity was measured at various concentrations with fixed substrate and ATP concentrations in kinetic mode. Assays in kinetic mode allows for real-time assay optimization. Note that at higher enzyme concentrations the substrate is depleted and a plateau is reached as expected.
– (B.) PKA and JNK2α2 kinases were incubated with kemptide and ATF-2, respectively and their activities were titrated in the presence of 25 µM ATP. The ADP accumulation assay shows a high tolerance for ATP and produces a robust assay window. This allows the assay to be applicable for kinases with a broad range of activity and for the determination of compound activity at or above the ATP K_m.

Profile Kinase Inhibitors

Profiling protein kinase A (PKA) inhibitors. Staurosporine (SS) and H-7 inhibition of PKA (10 ng/mL) activity in the presence of 25 µM ATP. IC₅₀ values for staurosporine and H-7 for PKAα are consistent with the literature and demonstrate the ability of the assay to identify weak inhibitors (normalized as percentage signal change in endpoint mode).

Rank Order Kinase Activity in the Presence of an Inhibitor

Rank ordering of kinase activity. Staurosprine was titrated for three kinases (PKA, JNK2α2, and CK1) with peptide substrates using the endpoint procedure. Peptide substrates included 100 μM kemptide for PKA, 250 μM CK peptide for CK1, and 100 μM ATF2 peptide for JNK2α2, and ATP concentrations was held constant at their respective Km concentrations (25 μM for PKA, 50 μM for CK1, and 100 μM for JNK2α2). The assay correctly represents the rank order of activity of the three kinases (PKA > CK1 > JNK2α2) and suggests that staurosporine is a potent inhibitor of PKA and a weak inhibitor for both CK1 and JNK2α2.