Key words: AMARA • kinase assay • LEADseeker • chemiluminescence
It is predicted that at least 500 kinase and phosphatase enzymes exist, their activities having wide-ranging regulatory responses. Many kinases are part of a signaling cascade in disease pathways such as inflammation and cancer, so identifying modulators of kinase and phosphatase activity may provide new therapeutic agents.
This application note provides recommendations for the successful imaging of the AMARA HitHunter™ Chemiluminescence Kinase Assay on the LEADseeker™ Multimodality Imaging System and demonstrates the assay’s ability to measure the functional activity of serine/threonine kinase AMP in the phosphorylation of the AMARA substrate (AMARAASAAALARRR).
Reagents and instrumentation
AMARA HitHunter Chemiluminescence 90-0060-02
Kinase Assay Kit*, 800 (384 wells)†
LEADseeker Multimodality Imaging System 18-1140-71
[14C] Uniform Reference Plate CFQ11387-4UCI
Additional materials required
AMP-activated protein kinase (Upstate #14-305)
ATP (Sigma A-7699)
Adenosine monophosphate (AMP) (Sigma A-2002)
384-well white flat bottom polystyrene (Corning 3705)
not treated microplates
Graphpad Prism software v.4 (GraphPad Software)
A flat field correction was established using a [14C] Uniform Reference Plate. No background correction was required.
Plates were imaged on the LEADseeker Multimodality Imaging System for 60 s with coincident averaging and 3 × 3 binning.
1. The kinase enzyme was diluted using the antibody reagent supplied in the kit to give a range of working stocks yielding a final concentration range in the assay from 0.05 to 100 units/well.
2. The AMARA substrate and ATP were diluted with the assay buffer supplied in the kit to yield working stocks of 20-µM AMARA substrate, 40-µM ATP, and 400-µM AMP.
3. The chemiluminescent substrate (CLS) was prepared by mixing 1 part Galacton-Star™, 5 parts Emerald-II™, and 19 parts CLS diluent.
4. The enzyme donor (ED)/CLS mix was prepared by carefully mixing 1 part ED reagent with 1 part CLS and was used within 6 h.
5. The enzyme acceptor (EA) reagent was used as supplied.
1. To the kinase assay wells, add 10 µl of kinase reaction buffer to each well. To the standard curve wells, add 10 µl of AMARA standard and 10 µl of AMARA antibody reagent.
2. Incubate plate in darkness for 60 min at room temperature (20–25 ºC).
3. Add 20 µl of ED/CLS to each well.
4. Incubate plate in darkness for 60 min (± 15 min) at room temperature.
5. Add 10 µl of EA reagent to each well.
6. Incubate plate in darkness at room temperature for at least 60 min or overnight.
7. 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.
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 hosphate (AMP).
† Other pack sizes available, please inquire for details.
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