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The β-Gal ELISA is used to quantitatively measure β-Gal expression in eukaryotic cells that are transfected with a plasmid bearing a β-Gal-encoding reporter gene.

• Standardized: Allows direct comparison of data from different sets of experiments, even when kits from different production lots are used
• More sensitive: More sensitive than colorimetric β-Gal assays
• More accurate: Measures the actual amount of β-Gal protein synthesized, not just β-Gal enzyme activity
• More specific: Detects bacterial, not endogenous β-Gal
• Fast, approximately 4 hours elapse from start to finish
• Easy to perform: Follows a standard ELISA protocol

Assay time: approx. 4 hours
Sample material: Cell extracts
Sensitivity:≥30 pg/ml (6 pg/well)
Specificity: The test is specific for β-Gal (β-D-galactoside galactohydrolase, EC 3.2.1.23) from E. coli. It does not crossreact with eukaryotic, lysosomal β-galactosidase.
Standard: The β-Gal enzyme from E. coli, included in the kit for the purpose of compiling a standard calibration curve, is provided with lot-specific content data as determined by immunoassay.

Promoter activity in transfected mammalian cells is generally studied by linking the promoter sequence to a gene that encodes an easily detectable “reporter” protein, such as chloramphenicol acetyltransferase (CAT), β-galactosidase (β-Gal), or luciferase (Luc).
The E. coli lacZ gene, encoding the enzyme β-galactosidase (β-Gal), has become one of the standard reporter genes used in transfection experiments. Traditionally, β-Gal activity is measured using colorimetric assays with o-nitrophenol-β-D-galactopyranoside (ONPG) or chlorophenol red β-D-galactopyranoside (CPRG) as substrates. These assays are not sensitive enough to detect small amounts of the enzyme. Another disadvantage is that in order to enable discrimination between the endogenous lysosomal β-Gal activity and the bacterial enzyme, the colorimetric assays must be performed at an alkaline pH. In contrast, β-Gal ELISA specifically detects the bacterial enzyme, but not the analogous lysosomal β-galactosidase.

1. β-Gal Enzyme (from E. coli)
2. Anti-β-Gal-digoxigenin
3. Anti-DIG-peroxidase
4. POD Substrate
5. Substrate Enhancer
6. Washing Buffer concentrate, 10x
7. Sample Buffer
8. Lysis Buffer concentrate, 5x
9. Two Microplates, strip frame with 12 modules of 8 wells, precoated with anti-β-Gal
10. Self-adhesive Plate Cover Foils (3)

The β-Gal ELISA is based on the sandwich ELISA principle. Following lysis of the transfected cells, the cell extracts (containing the β-Gal enzyme) are added to the wells of the microplate, which are precoated with a polyclonal antibody to β-Gal (anti-β-Gal). All β-Gal contained in the cell extracts binds to the anti-β-Gal antibody that is bound to the microplate surface. Next, a digoxigenin-labeled antibody to β-Gal (anti-β-Gal DIG) is added, then bound to the β-Gal. In the next step, an antibody to digoxigenin conjugated to peroxidase (anti-DIG-POD) is added, then bound to digoxigenin. In the final step, the peroxidase substrate (ABTS) is added. The peroxidase enzymes catalyze the cleavage of the substrate to produce a colored reaction product. Absorbance of the samples is determined using an ELISA reader, and is directly correlated to the level of β-Gal present in the cell extract. The sensitivity of the assay can be enhanced using ABTS with a substrate enhancer.

Figure 1: Test principle.

Figure 2: A typical calibration curve using ABTS without or with substrate enhancer as the peroxidase substrate.

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