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Performance of the AMARA HitHunter Chemiluminescence Kinase Assay with the LEADseeker Multimodality Imaging System

Seal plate and image on the LEADseeker Multimodality Imaging System for between 10 and 60 s.

Preparation of standard curve
The standard curve was established by preparing twelve 3-fold serial dilutions (using assay buffer) of the phosphorylated AMARA standard supplied in the kit. The final concentration range of the standard curve was 5.6 × 10-11 to 1.0 × 10-5 M in a final volume of 20 µl/well. Each standard concentration was assayed in triplicate.

AMP kinase titration procedure
AMP kinase was diluted in antibody reagent to yield a final concentration range of 0.05 to 100 mU/well. Each test point was assayed in triplicate. The AMARA substrate was used at a final concentration of 10 µM; ATP was used at a final concentration of 20 µM and AMP at 200 µM. The reaction was incubated for 60 min at room temperature. Following incubation with the detection reagents, the reaction was imaged on the LEADseeker Multimodality Imaging system for 60 s.


Conclusion
The AMARA HitHunter Chemiluminescence Kinase Assay has been successfully used on the LEADseeker Multimodality Imaging System and is suitable for measuring the phosphorylation of AMARA substrate by serine/threonine kinase AMP.


* The assay buffer may require supplementation with cofactors, which will be enzyme specific. This application note describes how the AMARA HitHunter™ Chemiluminescence Kinase Assay performs with serine/threonine kinase AMP; no additional cofactors were added to the assay buffer for this enzyme; however, there is a 3-fold increase in activation of the enzyme in the presence of adenosine 5’-monop
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