|
The commonly used method of eukarYOtic gene expression regulation research is the detection of reporter genes, and bioluminescence is the most commonly used and effective means of reporter gene detection. Luciferase can catalyze the conversion of the substrate luciferin and emit photons. This product provides a rapid, sensitive and stable detection method for the expression of firefly luciferase reporter gene in mammalian cells. It can detect the lowest luciferase of 10-20 mol, and has a good linear relationship in the concentration range of 10-14 to 10-20mol. Component： 
Instruction：
1. Cell lysis
( 1 ) The medium in the cells transfected with the reporter gene was removed and gently washed with PBS ( adherent cells can be directly subjected to this operation, and suspended cells should be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. 
Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ).
( 2 ) The pyrolysis products after full pyrolysis were centrifuged at 10000-15000 rpm for 3-5 min, and the supernatant was collected.
2.Working fluid configuration
( 1 ) Restore all components to room temperature.
( 2 ) The component C ( storage solution ) was fully diluted with component B to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing.
Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use.
3.chemiluminescence value detection
( 1 ) According to the instructions of the instrument, open the instrument with chemiluminescence detection function, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s.
( 2 ) The cell lysis products were added to the measuring tube according to the volume of 20 ~ 100 μL ( keep the same amount of samples each time ). 1 × Lysis Buffer was blank control.
( 3 ) 100 μL firefly luciferase detection solution was added to determine RLU ( Relative light unit ) ( Shaking mixing function is recommended for microplate reader ).
Matters needing attention： 1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments. 2.Temperature will affect the activity of the enzyme, so all reagents should be placed at room temperature before use. 3.If the single-tube fluorescence detector is used, the time from the mixing of each sample with the determination reagent to the time before the determination should be consistent. 4.The strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 5.In order to prevent interference between holes, it is recommended to use a white opaque hole plate. 6.For your safety and health, please wear experimental clothes and wear disposable gloves.
Product characteristic： 1.Rapid : Rapid : Cell lysis was completed within 10-15 min ; 2.Convenience : the reagent is easy to prepare, the sample detection steps are simple ; 3.Sensitive : capable of detecting a minimum of 10-20 mol luciferase ; 4.the linear range of enzyme concentration can reach 8 orders of magnitude. Recommendation： Component C is recommended to use sterile water in advance to prepare 2 mg / mL stock solution.B component and C component configured as storage solution, and small batch packing according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing.
Scope of application：
Study on gene expression regulation and promoter |
C2518622