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ARC-Photo Fluorescence Polarization Kinase Binding Assay Kit
Flyer in pdf

Screening and characterization of inhibitors of protein kinases (PK) is usually performed in the form of kinetic studies where the retarding effect of the compound on the rate of kinase-catalyzed phosphorylation reaction is assigned. Alternatively, kinase inhibitors may be identified by direct or indirect measurement of binding of the compound to the kinase.

The kit ARC-Photo is applicable in competition displacement assays for identification and characterization of inhibitors of many protein kinases and determination of the active (binding) concentration of a protein kinase. The fluorescent probe ARC-Photo has very high affinity towards several kinases (e.g., Kd < 1 nM for cAMP-dependent protein kinase, PKA). The bisubstrate-analog character of the probe (the inhibitor ARC simultaneously binds to both pockets of PK) enables the characterization of inhibitors (and substrates) targeted to ATP binding site and/or substrate protein/peptide binding domain of the kinase.

As tested in a panel of 52 protein kinases ARC-type compounds are good inhibitors of basophilic PK (neighborhood of phosphorylation sites of substrates is rich in arginine and/or lysine residues). Thus ARC-Photo is a potential fluorescent probe for many kinases with such qualities (e.g., majority of PK of the AGC group are basophilic).

Key features of the kit:
Fast Results in 30-60 minutes
Easy Just mix and read fluorescence anisotropy, no need for substrates, antibodies or ATP; no separation procedures and radioactive materials used
Multi-porpose Can be used for determination of the concentration of the kinase, screening of inhibitors, and determining the affinity of inhibitors and substrates
Comprehensive Competitive inhibitor binding to kinase active site is measured which enables the determination of affinities of ATP- and protein-competitive ligands of basophilic protein kinases
Economic Low concentrations of the probe (< 5 nM) and the kinase (< 5 nM) and minute reaction volumes (< 30 microL in the 384-well microtiter plate format) are required
Productive Easy to test with new kinases (incl. mutated and non-active forms of PK) as no substrates and product-associated antibodies are needed

ARC-Photo is a fluorescent probe with excellent characteristics:
High chemical and photo-stability, soluble in water;
Long wave-length fluorescence (540 nm excitation and 590 nm emission with
    20 nm bandpass filters);
Large signal window (> 130 mA) ensures high Z' factors of > 0.7.

Compare ARC-Photo with other available products on the market!

Mechanism of ARC-Photo

1. ARC-Photo is bound to protein kinase.

2. Bisubstrate character of ARC means that ARC-Photo is displaced from its complex by both ATP and protein binding-site targeted inhibitors (and substrates).

3. The displacement of ARC-Photo (MW<2000) from its slow-rotating complex with the kinase leads to substantial decrease of the fluorescence anisotropy of the solution which can be measured on a microplate reader with fluorescence polarization detection.