D-fluorescein potassium salt use method and FAQ
D-fluorescein potassium salt is a water-soluble substrate for luciferase and is present in a variety of luminescent organisms. Under the catalysis of ATP and luciferase, D-luciferin potassium is oxidized to produce blue-green light (560 nm). When the substrate is excessive, the photon number produced is positively correlated with the concentration of luciferase. The Luc gene encoding luciferase is a commonly used reporter gene for plants, bacteria, and mammalian cells. Luciferase down to a level of 0.02 pg can be easily detected due to no background interference.
D-luciferin potassium salt can express the expression of luc marker gene and luciferase fusion protein in living cells, tissues and organisms, and is widely used in reporter gene analysis, immunoassay and health monitoring.
Chemical name: 4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt
CAS number: 115144-35-9
Molecular formula: C11H7N2O3S2K
Molecular weight: 318.42 g/mol
Purity: 99.7 % (HPLC)
application
In vitro fluorescence detection: preparation of fluorescein
Reagents:
D-fluorescein potassium salt (Gold Biotechnology catalog ID: LUCK)
Molecular biology water
Medium
Steps:
1. Prepare a 30 mg/L (200×) stock solution with molecular biology grade water.
a. Repeat the trip until the fluorescein is completely dissolved.
b. It can be used now or stored at -20 °C.
2. Add the prepared fluorescein storage solution to the pre-warmed tissue culture medium to make the final concentration of fluorescein: 150ug/ml.
3. Aspirate the cultured cells (no media remaining).
4. Add 150 ug/ml fluorescein solution to the cells immediately prior to imaging.
a. To enhance the signal, the cultured cells can be incubated briefly at 37 °C prior to imaging.
In vivo fluorescence detection in mice: preparation of fluorescein
Reagents:
D-fluorescein potassium salt (Gold Biotechnology catalog ID: LUCK)
DPBS does not contain Mg2+ and Ca2+
Steps:
1. The final concentration of lysin in DPBS was: 15 mg/ml.
2. Filter the solution with a 0.2um filter.
3. Each mouse was injected with 10 ul (15 mg/ml) of fluorescein stock solution per gram of body weight.
4. Inject the mouse into the abdominal cavity within 10-15 minutes before imaging.
a. Fluorescence kinetic studies were applied to each animal model to determine peak signal time.
Answers to common questions about D-fluorescein potassium salt:
1. Can fluorescein be used in living animals?
A: Many of our customers use fluorescein to work on animals. Currently, there are two major manufacturers of synthetic fluorescein in the world, so you are currently using one of these two manufacturers. Please note: The fluorescein sold by Promega is Beetle Luciferin, and our company sells Firefly Luciferin, but the structure of the two is the same (the structural formula is as follows).
2. How to dissolve fluorescein? How to make it stable after dissolution?
A: Many customers have told us that fluorescein is soluble in water and then stored in -80 ° C, without significant impact. However, there are also contradictory reports on the stability of fluorescein after dissolution. For most sensitive experiments, in order to reduce the variables in some experiments, we strongly recommend that you use it now (such as low concentrations of enzymes or inappropriate reaction temperatures or inappropriate salt concentrations, etc. may require high concentrations of substrate To drive the completion of the reaction.)
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