Reporter gene assay β-galactosidase substrates - SK-Hep-1

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.

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Found 3 matching solutions for this experiment

Protocol tips
Add 150µl of Assay 2X Buffer to sample.

Incubate the reactions at 37°C for 30 minutes or until a faint yellow color has developed.
Downstream tips
Read the absorbance at 420nm.
Protocol tips
Add 1 ml of the β-Galactosidase Staining Solution and incubate the plate at 37°C at least overnight in a dry incubator (no CO2)
Protocol tips
Add 1 ml of the β-Galactosidase Staining Solution and incubate the plate at 37°C at least overnight in a dry incubator (no CO2)
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