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D-Luciferin

CAS No. : 2591-17-5

MCE 国际站：D-Luciferin

产品活性：D-Luciferin 是荧光素酶 (Luc) 的天然底物，对萤火虫产生典型的黄绿光进行催化。该反应产生的 560 nm 化学发光在几秒钟内达到峰值，当底物荧光素过量时，光输出与荧光素酶浓度成正比。荧光素酶 (luc) 基因是研究和活性分子筛选的常用报告基因。化学发光技术实际上是无背景的，使得 luc 报告基因成为检测低水平基因表达的理想选择。在标准荧光计数仪中可以可靠地测量到 0.02 pg 的荧光素酶。除了用于基因表达的外，荧光素酶还用于 ATP 的检测。MCE 提供萤火虫荧光素酶 (HY-P1004) 、荧光素游离酸 (HY-12591A) 及其水溶性钠盐 (HY-12591) 和钾盐 (HY-12591B) 。

研究领域：Others

作用靶点：Fluorescent Dye

结构分类：其他类

In Vitro: 1. Precautions
a) The D-luciferin is readily soluble in aqueous buffers (pH 6.1-6.5) up to 100 mM. Stock solutions can be made in ATP-free water and stored at -20℃, protect from light. The free acid must be neutralized with an appropriate base to solubilize. 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.
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.
2.1 Example protocol for in vitro bioluminescent image assays
a) 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℃, 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 Luciferin working solution to cells, and incubate the cells for 5-10 minutes at 37℃ just prior to imaging.
2.2 Example protocol for in vivo bioluminescent image assays
a) Prepare a 15 mg/mL Luciferin stock solution in DPBS, without Mg²⁺ and Ca²⁺. Mix well.
b) Filter sterilizes the solution through a 0.2 μm filter. Use immediately, or make single use aliquots, and store at -20℃, avoid freeze-thaw cycles, avoid exposure to the light.
c) Inject the luciferin intra-peritoneally (i.p.) 10-15 minutes before imaging at 150 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.
2.3 Example protocol for luciferin reporter assays
a) Prepare a 100 mM Luciferin stock solution in sterile water. 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 luciferin working solution according to manufacturer’s instructions.
e) Inject 200 μL of luciferin working solution with no delay and a 10 second integration time.

In Vivo: Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and D-luciferin as a substrate is currently the most widely employed technique. The total signal intensity is plotted against the time after D-luciferin injection to generate a time-intensity curve. In addition to the peak signal, the signals at fixed time points (5, 10, 15, and 20 min) after D-luciferin injection are determined as alternatives to the peak signal. The signal in a given time-intensity curve is normalized for the peak signal in the curve to represent the pattern of temporal changes after D-luciferin injection.
Inject with 10 μL of D-luciferin (intraperitoneally or intravenously) stock solution per gram of body weight: normally ~200 μL for a 20 g mouse for a standard 150 mg/kg injection.
Thaw D-Luciferin (either Potassium or Sodium Salt) at room temperature and dissolve in dPBS (no calcium or magnesium) to a final concentration of 15 mg/mL. Pre-wet a 0.22 μm filter by drawing through 5-10 mL of sterile H₂O and discard water. Sterilize the D-Luciferin solution through the prepared 0.22 μm syringe filter.

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