The non-enzymatic synthesis of luciferin from cysteine and p-benzoquinone (1:1), reported by Kanie et al. was replicated in this study using purified A. vivianii firefly luciferase (AmyLuc) and 4 mM ATP in 90 mM Tris–HCl pH 7.5 buffer and 5 mM MgSO4 to sense the formation of D-luciferin. For this purpose, reactions different compounds were mixed and analyzed in an Elisa plate. The following treatments were tested: (1) Buffer + MgATP; (2) Buffer + MgATP + AmyLuc; (3) Buffer + MgATP + AmyLuc + d -cys + BQ; (4) Buffer + MgATP + AmyLuc + d -Cys; (5) Buffer + MgATP + AmyLuc + l -cys + BQ; (6) Buffer + MgATP + AmyLuc + l -cys; (7) Buffer + MgATP + AmyLuc + l -cys; (8) Buffer + MgATP + AmyLuc + d -cys + Hydroquinone; (9) Buffer + MgATP + AmyLuc + l -cys + Hydroquinone; (10) Buffer + MgATP + AmyLuc + Hydroquinone. The luciferin formation was followed by bioluminescence using Berthold Nightowl CCD camera with images taken every 15 min, and also confirmed by detection of fluorescence upon irradiation by UV light on the Elisa plate. The assays were performed in triplicate.
Nightowl ccd camera
Manufactured by Berthold Technologies
The Nightowl CCD camera is a high-performance scientific imaging device designed for low-light applications. It features a charge-coupled device (CCD) sensor that captures images with high sensitivity and resolution. The core function of the Nightowl CCD camera is to provide reliable and accurate image data for various scientific and research purposes.
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2 protocols using nightowl ccd camera
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Firefly Luciferin Synthesis Assay
2
Non-enzymatic Luciferin Synthesis Assay
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The non-enzymatic synthesis of luciferin fromcysteine and p-benzoquinone (1:1), reported by Kanie et al. (2016) was replicated in this study using purified A. vivianii firefly luciferase (AmyLuc) and4 mM ATP in 90 mM Tris-HCl pH 7.5 buffer to sense the formation of D-luciferin.
For this purpose, reactions different compounds were mixed and analyzed in an Elisa plate. The following treatments were tested: 1) Buffer + ATP; 2) Buffer + ATP + AmyLuc; 3) Buffer + ATP + AmyLuc + D-cys + BQ; 4) Buffer + ATP + AmyLuc + D-Cys; 5) Buffer + ATP + AmyLuc + L-cys + BQ; 6) Buffer + ATP + AmyLuc + L-cys; 7) Buffer + ATP + AmyLuc + L-cys; 8) Buffer + ATP + AmyLuc + D-cys + Hydroquinone; 9) Buffer + ATP + AmyLuc + L-cys + Hydroquinone; 10) Buffer + ATP + AmyLuc + Hydroquinone. The luciferin formation was followed by bioluminescence using Berthold Nightowl CCD camera with images taken every 15 minutes, and also confirmed by detection of fluorescence upon irradiation by UV light on the Elisa plate. The assays were performed in triplicate.
For this purpose, reactions different compounds were mixed and analyzed in an Elisa plate. The following treatments were tested: 1) Buffer + ATP; 2) Buffer + ATP + AmyLuc; 3) Buffer + ATP + AmyLuc + D-cys + BQ; 4) Buffer + ATP + AmyLuc + D-Cys; 5) Buffer + ATP + AmyLuc + L-cys + BQ; 6) Buffer + ATP + AmyLuc + L-cys; 7) Buffer + ATP + AmyLuc + L-cys; 8) Buffer + ATP + AmyLuc + D-cys + Hydroquinone; 9) Buffer + ATP + AmyLuc + L-cys + Hydroquinone; 10) Buffer + ATP + AmyLuc + Hydroquinone. The luciferin formation was followed by bioluminescence using Berthold Nightowl CCD camera with images taken every 15 minutes, and also confirmed by detection of fluorescence upon irradiation by UV light on the Elisa plate. The assays were performed in triplicate.
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