Uses
For research use only. Not for use in diagnostic procedures.
The β-Gal ELISA is used to quantitatively measure β-Gal expression in eukaryotic cells transfected with a plasmid bearing a β-Gal-encoding reporter gene. The β-Gal ELISA may also be used for quantification of fusion proteins, produced by in-frame cloning into an appropriate β-Gal encoding DNA construct.
General Description
β-Gal (β-D-galactoside galactohydrolase, EC 3.2.1.23) from
E. coli is specifically detected by the β-Gal ELISA. 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.
Contents:
- β-Gal Enzyme (from E. coli)
- Anti-β-Gal-digoxigenin
- Anti-DIG-peroxidase
- POD Substrate
- Substrate Enhancer
- Washing Buffer concentrate, 10x
- Sample Buffer
- Lysis Buffer concentrate, 5x
- Two Microplates, strip frame with 12 modules of 8 wells, precoated with anti-β-Gal
- Self-adhesive Plate Cover Foils (3)
Biochem/physiol Actions
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.
