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

Software)


Method
LEADseeker setup
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.

Reagent preparation
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.

Assay procedure

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.
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