D-Luciferin

D-luciferin is the natural substrate of the enzyme luciferase (Luc) that catalyzes the production of the typical yellowgreen light of fireflies. The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase concentration when the substrate luciferin is present in excess. The luciferase (luc) gene is a popular reporter gene for research and agent screening. Chemiluminescent techniques are virtually background-free, making the luc reporter gene ideal for detecting low-level gene expression. As little as 0.02 pg of luciferase can be reliably measured in a standard scintillation counter. In addition to its role as a reporter of gene expression, luciferase is commonly used in an extremely sensitive assay for ATP^[1]. We of er the firefly luciferase (HY-P1004), luciferin free acid (HY-12591A), as well as its water-soluble sodium salts (HY-12591) and potassium salts (HY-12591B) . In Vitro:1. Precautions
a) There are three forms of D-luciferin, namely free acid (HY-12591A), potassium salt (HY-12591B) and sodium salt (HY-12591). The sodium salt and potassium salt forms of D-luciferin are easily soluble in aqueous buffers (pH 6.1-6.5). Stock solutions can be made in ATP-free water and stored at -20℃, protect from light. The free acid can be neutralized with DMSO or an appropriate base to dissolve. At a higher pH, luciferin undergoes a base-catalyzed formation of dehydroluciferin, as well as racemization to the L-isomer.
b) The D-luciferin can be used with any existing reporter assay or ATP assay system.
c) 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.
2. Experimental Protocols
This protocol only provides a guideline, and should be modified according to your specific needs.
2.1 Example protocol for in vitro bioluminescent image assays
a) Prepare D-luciferin stock solution in DMSO. Mix well. Use immediately, or make single use aliquots, and store at -20℃, avoid freeze-thaw cycles, avoid exposure to the light.
b) Prepare a 0.5-1 mM working solution of D-Luciferin in pre-warmed tissue culture medium.
c) Aspirate media from cultured cells.
d) Add D-Luciferin working solution to cells, and incubate the cells for 5-10 minutes at 37℃ just prior to imaging.
2.2 In vivo assays
Note: Since the in vivo experimental dose is relatively large, generally 150 mg/kg, it is recommended to choose HY-12591B or HY-12591.
2.3 Example protocol for D-Luciferin reporter assays
a) Prepare D-luciferin stock solution in DMSO. Use immediately, or make single use aliquots, and store at -20℃, avoid freeze-thaw cycles, avoid exposure to the light.
b) 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.
c) Pipette 5-10 μLof cell lysate into a microplate. Use lysis reagent or buffer without lysate as a blank.
d) Prime luminometer with D-Luciferin working solution according to manufacturer’s instructions.
e) Inject 200 μL of D-luciferin working solution with no delay and a 10 second integration time.