D-Luciferin, sodium salt *CAS#: 103404-75-7*

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Chemical structure for D-Luciferin, sodium salt *CAS#: 103404-75-7*
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Ex/Em (nm)328/533
CAS #103404-75-7
Storage Freeze (<-15 °C)
Minimize light exposure
Category Cell Biology
Reporter Gene Enzymes
Related Secondary Reagents
Luciferin is the most popular and versatile bioluminescent substrate. The firefly luciferase/luciferin bioluminescent system is found in the firefly (Photinus pyralis) and several other beetles. Luciferase oxidizes ATP-activated luciferin through a dioxetanone intermediate. Firefly luciferase produces light by the ATP-dependent oxidation of luciferin. The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase activity when luciferin and ATP are present in excess. Firefly luciferase has long been conjugated to antibodies and used as a label in immunoassays using luciferin as the substrate for detection. Compared to HRP and alkaline phosphatase, luciferase is less tolerant to chemical modifications. One particular advantage to the enzyme is that there is low endogenous luciferase activity in mammalian tissues besides its high sensitivity. Another important use of luciferase is in the area of hygiene monitoring. The luciferase/luciferin system can be used to detect contamination because ATP, present in all living organisms, is required to produce luminescence. The main application for this type of ATP bioluminescence is quality assurance by testing surfacesin food processing plants to determine whether or not there iscontamination of eitherequipment or products.

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of D-Luciferin, sodium salt *CAS#: 103404-75-7* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =


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This protocol only provides a guideline, and should be modified according to your specific needs.

Note1: The D-luciferin salts are readily soluble in aqueous buffers up to 100 mM. Stock solutions can be made in ATP-free water and stored at -20°C, protect from light. The free acid must be neutralized with an appropriate base to solubilize.

Note2: The D-luciferin can be used with any existing reporter assay or ATP assay system.

Note 3: If testing for ATP, minimize all possible sources of ATP contamination by wearing gloves and using ATP-free containers. Use only sterile ATP-free water and reagents. Use autoclaved water for all reagent preparations.


The following protocol is an example for potassium and sodium salt preparation, it can be adapted for most cell types and in vivo animal use.

1.       Example protocol for in vitro bioluminescent image assays

1.1.   Prepare a 100 mM (100-200X) Luciferin stock solution in sterile water. Mix well. Use immediately, or make single use aliquots, and store at -20 °C, avoid freeze-thaw cycles, avoid exposure to the light.

1.2.   Prepare a 0.5-1 mM working solution of D-Luciferin in pre-warmed tissue culture medium.

1.3.   Aspirate media from cultured cells.

1.4.   Add Luciferin working solution to cells, and incubate the cells for 5-10 minutes at 37 °C just prior to imaging.


2.       Example protocol for in vivo bioluminescent image assays

2.1.   Prepare a 15 mg/mL Luciferin stock solution in DPBS, w/o Mg2+ and Ca2+. Mix well.

2.2.   Filter sterilizes the solution through a 0.2 μm filter. Use immediately, or make single use aliquots, and store at -20 °C, avoid freeze-thaw cycles, avoid exposure to the light.

2.3.   Inject the luciferin intra-peritoneally (i.p.) 10-15 minutes before imaging at150 mg/kg (or 10 μL/g of luciferin stock solution) of the animal body weight.

Note: A kinetic study of luciferin should be performed for each animal model to determine peak signal time.


3.       Example protocol for luciferin reporter assays

3.1.   Prepare a 100 mM Luciferin stock solution in sterile water. Use immediately, or make single use aliquots, and store at -20 °C, avoid freeze-thaw cycles, avoid exposure to the light.

3.2.    Prepare a 1 mM working solution of D-Luciferin with 3 mM ATP, 1 mM DTT and 15 mM MgSO4 in 25 mM tricine buffer pH 7.8.

3.3.   Pipette 5-10 μl of cell lysate into a microplate. Use lysis reagent or buffer without lysate as a blank.

3.4.   Prime luminometer with luciferin working solution according to manufacturer’s instructions.

3.5.   Inject 200 μl of luciferin working solution with no delay and a 10 second integration time.

References & Citations

C3-Luc Cells Are an Excellent Model for Evaluation of Cellular Immunity following HPV16L1 Vaccination
Authors: Li-Li Li, He-Rong Wang, Zhi-Yi Zhou, Jing Luo, Xiao-Li Wang, Xiang-Qian Xiao, Yu-Bai Zhou, Yi Zeng
Journal: PloS one (2016): e0149748

Genome-wide microRNA analysis identifies miR-188-3p as novel prognostic marker and molecular factor involved in colorectal carcinogenesis
Authors: Martin Pichler, Verena Stiegelbauer, Petra Vychytilova-Faltejskova, Cristina Ivan, Hui Ling, Elke Winter, Xinna Zhang, Matthew Goblirsch, Annika Wulf-Goldenberg, Masahisa Ohtsuka
Journal: American Association for Cancer Research (2016): clincanres--0497

Identification of a Novel Protein Kinase A Inhibitor by Bioluminescence-Based Screening
Authors: Tetsuya Ishimoto, Kenji Azechi, Hisashi Mori
Journal: Biological and Pharmaceutical Bulletin (2015): 1969--1974

Discovery of novel adenylyl cyclase inhibitor by cell-based screening
Authors: Hiroki Mano, Tetsuya Ishimoto, Takuya Okada, Naoki Toyooka, Hisashi Mori
Journal: Biological and Pharmaceutical Bulletin (2014): 1689--1693

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