2. Mono-Lumi Firefly Luciferase Reporter Gene Assay Kit, 100T $198

Product Information

Description

Firefly-luciferase system is commonly used for reporter gene detection. Reporter gene plasmid was constructed by cloning the transcriptional regulatory elements of the target gene into the upstream and downstream of luciferase. The plasmids were then transfected into cells, and after treatment with drugs or other stimulants, the cells were harvested and lysed to measure luciferase activity in the lysates. Luciferase activity was used to determine the transcriptional regulation of target genes by exogenous stimuli such as drugs.

The Mono-Lumi Firefly Luciferase Reporter Gene Assay Kit has high sensitivity, wide detection range and stable fluorescence signal. Firefly Luciferase was used to catalyze the oxidation of Firefly luciferin substrate to oxyluciferin under the conditions of Mg2+, ATP, and O2, and generate bioluminescence in this process. Therefore, firefly luciferase activity can be rapidly detected.

Storage and Handling Conditions

Transport with dry ice; Store at -80℃ away from light, valid for at least one year; Store at -20℃ away from light, recommended for use within 6 months.

Component

Assay Protocol / Procedures

1. Preparation of 1× firefly luciferase reaction working solution: 

1.1 Each kit group is thawed at room temperature and mixed upside down to ensure that each component is completely dissolved. Luciferase Reaction Substrate (50×) shall be melted on ice and centrifuged for a short time to ensure that the reagents sink to the bottom of the tube;

1.2 Luciferase Reaction Substrate (50×) diluted 50 times with Luciferase Reaction Buffer; 10 μL Luciferase Reaction Substrate (50×) +490 μL Luciferase Reaction Buffer were mixed to obtain 500 μL 1× firefly Luciferase Reaction working solution for later use. Pay attention to the dosage of preparation, avoid waste. 1× firefly luciferase reaction working solution is recommended to be prepared and used. If stored at -20℃, use within one month and avoid repeated freezing/thawing.

2. Cell pretreatment and lysis:

2.1 The cells were planted in the corresponding well plates in advance, and transfected or other relevant pretreatment was carried out according to the experimental needs;

2.2 For adherent cells: After removing the original cell medium, add the corresponding amount of cell lysate according to the table below to cover the cells; For the suspended cells, transfer the cells to a centrifuge tube, remove the culture medium by centrifugation, and resuspended the cells by adding the corresponding amount of cell lysate according to the table below.

2.3 After lysis at room temperature for 10-20 min, the cells were scraped and collected in a 1.5ml centrifuge tube, centrifuged at 12000 g at 4℃ for 10 min, and the supernatant (cell lysate) was kept for later use.

3. Firefly luciferase detection:

3.1 The 1× firefly luciferase reaction liquid prepared in advance was balanced to room temperature and added to the light-tight white 96-well plate at 100 μL/ well;

3.2 The cell lysis supernatant of step 2 was added to the above 96-well plate at 20 μL/well;

3.3 When the horizontal shock is uniform, the chemiluminescence detection is performed immediately with Luminometer, multifunctional microplate reader with chemiluminescence detection module or other instruments capable of detecting bioluminescence.

Note

1. Before testing, the 1× firefly luciferase reaction working solution prepared in advance should be restored to room temperature before use.

2. After thawing, Luciferase Reaction Buffer has some precipitation, which is a normal phenomenon. Shake thoroughly before use to ensure complete dissolution.

3. The 1× luciferase reaction working solution is prone to oxidation reaction. For multi-sample detection, the addition of 1× firefly luciferase reaction working solution should be controlled in a short time as far as possible. It is recommended to use the multichannel pipettes to add samples, and pay attention to whether the suction volume of each hole of the gun is consistent.

4. To prevent interference between holes, it is recommended to use an opaque white hole plate. At the same time, the opaque black orifice plate can also be used, but the black will absorb the fluorescence signal, reducing the fluorescence signal of detection.

5. For your health and safety, please wear laboratory coat and gloves when operating.