Product Catalog

The β-Gal Reporter Gene Assay, chemiluminescent, is used to quantitatively measure β-Gal expression in eukaryotic cells that are transfected with a plasmid that bears the β-Gal-encoding lacZ gene as a reporter.

• Sensitive: Chemiluminescence technology allows detection of approximately 20 fg β-galactosidase in cell extracts
• High dynamic measuring range: Linear range over four to five orders of magnitude
• Constant light emission: The assay produces a long-lasting light emission instead of a short-peak kinetic
• Safe: No radioactive isotopes are used
• Fast: Approx. 1.5 to 2.5 hours elapse from start to finish
• Convenient: Kit contains all reagents needed, including lysis buffer, protease inhibitors, and a positive control

Assay time: 90 to 150 min
Detection range and sensitivity: Under assay conditions, the detection range for the positive control is between 20 fg and 20 ng (Figure 2). However, the exact detection limit and measuring range depend on the measuring device and the measuring conditions used.
Sample material: Cell extracts
Specificity: The β-Gal Reporter Gene Assay is designed for specifically measuring bacterial β-galactosidase activity. To achieve this, the enzyme reaction is conducted at a pH that is optimized for bacterial β-Gal, but allows no significant endogenous eukaryotic β-galactosidase activity. However, if very high endogenous β-galactosidase activity is found (e.g., with hepatic cells), a heat treatment can be performed to ensure the specific determination of the amount of bacterial β-galactosidase that is encoded by the transfected plasmid.

Transcriptional activity of eukaryotic promoters is generally studied by linking an easily detectable reporter gene to the regulatory sequence of interest. The lacZ gene of E. coli encodes for β-galactosidase. When fused to eukaryotic promoters, it is one of the standard reporter genes. Bacterial β-galactosidase (β-Gal) consists of four identical subunits, each with a molecular weight of 116 kD, which can be directly detected in extracts from transfected cells. To achieve this, the enzymatic activity is traditionally measured with the colorimetric substrates ONPG (2-nitrophenyl-β-D-galactopyranoside) or CPRG (chlorophenol red-β-D-galactopyranoside). With chemiluminescent substrates based on 1,2-dioxetanes, the sensitivity for enzymatic detection of β-Gal is increased by several orders of magnitude compared to the colorimetric substrates. By using this innovative technology, the β-Gal Reporter Gene Assay allows the investigation of weak promoters by a highly convenient non-isotopic method.

1. β-Gal Substrate, 100x, Galacton Plus
2. Assay Buffer
3. Enhancer, Emerald II
4. Initiation Solution
5. Protease Inhibitors
6. Lysis Buffer, 5x
7. Positive Control, β-galactosidase (E. coli), lyophilizate

The outstanding sensitivity of this chemiluminescent reporter-gene assay results from the unique features of the dioxetane-class β-galactosidase substrate and the two-step assay principle:
1. Enzyme reaction: In the first step, the substrate Galacton Plus becomes deglycosylated by the enzymatic activity of the β-galactosidase contained in the sample. This incubation step is performed at pH 7.8, at which bacterial β-galactosidase is highly active. At this neutral pH, the cleaved dioxetane is protonated and will not produce a light signal. This stable intermediate accumulates during the reaction, and provides a light signal once the pH is adjusted higher.
2. Light reaction: The light reaction is initiated by shifting the pH to a value higher than 12. Due to this shift in pH, the activated intermediate becomes deprotonated and decomposes with the emission of light (475 nm). The presence of special polymeric enhancer substances improves the quantum yield of the chemiluminescent reaction. Under reporter-gene assay conditions, the light signal peaks within a second, then decays with a half-life (t_1/2) of approximately 10 minutes.

Figure 1: Test principle.

 

Figure 2: Kinetic of light reaction under reporter gene assay conditions. For details please see pack insert.

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