AffiASSAY® Dual Luciferase Reporter Assay Kit

AffiASSAY® Dual Luciferase Reporter Assay Kit

The AffiASSAY® Dual Luciferase Reporter Assay Kit is used to detect gene regulation by transfecting cells with a reporter plasmid and measuring the fluorescence intensity of Luciferin substrate to reflect the level of Luciferase expression. To achieve dual luciferase reporter gene detection, Firefly luciferase is detected with Luciferin as a substrate, and Renilla Iuciferase is detected with coelenterazine as a substrate, while inhibiting the activity of Firefly luciferase. Dual Luciferase Reporter Assay Kit can detect the expression of Luciferase regulated by gene elements sensitively and efficiently. Usually, the transcriptional regulatory element is cloned upstream of Firefly luciferase, or the 3'-UTR regulatory region is cloned downstream of firefly luciferase. The transfected cells are induced by corresponding stimulator and lysed to determine the luciferase activity. The stimulatory-inducing effect of the regulatory elements is evaluated by luciferase activity. Renilla Iuciferase acts as an internal reference for correcting transfection efficiency to eliminate differences in cell number and transfection efficiency between wells. Firefly luciferase catalyzes the emission of Luciferin at 560 nm, and Renilla luciferase catalyzes the emission of coelenterazine at 465 nm.

Components

Storage Conditions:

Store at -30 ~ -15℃ and transport at ≤0℃. Dissolved and dispensed Reagent: long-term storage at -85 ~ -65℃, or short-term storage at -30 ~ -15℃ for no more than one month; Stop & Reaction Buffer: long-term storage at 2 ~ 8℃.

Self-prepared Materials:

It is applicable for the detection of cell gene regulation.

Notes:

For research use only. Not for use in diagnostic procedures.

• When used for the first time, the Reaction Buffer II (Luciferase) should be poured into a brown dark bottle containing lyophilized Luciferase substrate.
• Mix thoroughly and dispense according to the needs of use and protect from light at -70℃. Mix 5 × Cell Lysis Buffer and ddH2O in a ratio of 1:4 before use, and place on ice for use.
• Dissolve Renilla Substrate in absolute ethanol. Please centrifuge briefly for the first use and carefully measure the volume of the solution in the tube. If the liquid volume is found to be significantly reduced, please add absolute ethanol to make up the volume before saving.
• Place Renilla Substrate on ice during use. Calculate the usage amount, mix an appropriate amount of Stop & Reaction Buffer and Renilla Substrate at a ratio of 50:1. Store at room temperature and protect from light for later use.
• The enzymatic reaction is sensitive to temperature, so the cell lysis solution and the detection substrate solution should be equilibrated to room temperature before the sample is applied for testing.