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11539426001 Roche

β-Gal ELISA

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Properties

usage   sufficient for 192 tests
mfr. no.   Roche
shipped in   wet ice
storage temp.   2-8°C

Description

Analysis Note

No cross-reaction with eukaryotic, lysosomal β-galactosidase.

Application

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.

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.

Features and Benefits

• 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

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)

Other Notes

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

Packaging

1 kit containing 10 components.

Principle

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.

Preparation Note

Working concentration: Transfer 40 to 100 ng of labeled RNA Molecular Weight Marker I per lane or 20 to 50 ng of RNA Molecular Weight Marker II or III per lane to produce clearly visible banding patterns.
Note: Taking the lane (slot) size of the agarose gel into account, load 100 ng for RNA marker I and 50 ng for RNA marker II or III.
Working solution: β-Gal Enzyme stock solution (solution 1)

Reconstitute the lyophilizate (bottle 1) in 0.5 ml double dist. water.
The resulting concentration is calculated using the lot specific information is described below.

Anti-β-Gal-DIG (solution 2)

Reconstitute the lyophilizate (bottle 2) in 0.5 ml double dist. water (final conc.: 50 μg/ml).

Anti-β-Gal-DIG, working dilution (solution 2a)

To prepare the working concentration, dilute the reconstituted anti-β-Gal-DIG solution (50 μg/ml) with sample buffer (solution 7) to a final conc. of 0.5 μg anti-β-Gal-DIG/ml sample buffer (e.g., 100 μl of reconstituted Anti-β-Gal-DIG solution 9.9 ml of Sample
buffer (bottle 7) for 50 wells). Prepare freshly before use! Do not store!

Anti-DIG-POD (solution 3)

Reconstitute the lyophilizate (bottle 3) in 0.5 ml double dist. water (final conc. 20 U/ml).
Note: Do not freeze! DO NOT add sodium azide as a preservative because it inhibits the activity of the peroxidase!

Anti-DIG-POD, working dilution (solution 3a)

To prepare Anti-DIG-POD, working dilution, dilute the reconstituted anti-DIG-POD solution (20 U/ml) with Sample buffer (solution 7) to a final conc. of 150 mU/ml (e.g., 75 μl of reconstituted Anti-DIG-POD solution 9.925 ml of Sample buffer (bottle 7) for 50 wells). Prepare freshly before use! Do not store!

ABTS substrate solution with enhancer (solution 5)

Add 1 mg of Substrate enhancer per ml of ABTS substrate solution (solution 4) and mix by stirring for 30 min at 15 to 25 °C.
Stable for only 4 h prepare immediately before use!

Washing buffer, 1x (solution 6)

To prepare a ready-to-use Washing buffer, mix 1 part of the Washing buffer 10× (bottle 6) with 9 parts of double dist. water.

Sample buffer (solution 7)

Ready-to-use solution. Mix thoroughly before use.

Lysis buffer, 1x (solution 8)

Mix 1 part of 5× Lysis buffer concentrate (bottle 8) with 4 parts of double dist. water.
Note: 1 ml of this solution is required per 6 cm culture dish.
Storage conditions (working solution): β-Gal Enzyme stock solution (solution 1)
1 week at 2 to 8 °C or 12 months at -15 to -25 °C
Anti-β-Gal-DIG (solution 2)
6 months at 2 to 8 °C or 12 months if stored in aliquots at -15 to -25 °C
Anti-β-Gal-DIG, working dilution (solution 2a)
Prepare freshly before use! Do not store!
Anti-DIG-POD (solution 3)
6 months at 2 to 8 °C
Anti-DIG-POD, working dilution (solution 3a)
Prepare freshly before use! Do not store!
POD substrate (solution 4)
Stable until the expiration date indicated on the kit if stored at 2 to 8 °C.
ABTS substrate solution with enhancer (solution 5)
Stable for only 4 h, prepare immediately before use!
Washing buffer, 1x (solution 6)
6 months at 2 to 8 °C
Sample buffer (solution 7)
Stable until the expiration date indicated on the kit if stored at 2 to 8 °C.
Lysis buffer, 1x (solution 8)
-15 to -25 °C or 3 months at 2 to 8 °C

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.

Specifications

Assay time: approximately 4 hours
Sample material: Cell extracts
Sensitivity: ≥30 pg/ml (6 pg/well)
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.

Kit component only

Description

Product #

Add to Cart

β-Gal Enzyme (from E. coli)    
Anti-β-Gal-digoxigenin antibody    
Anti-Digoxigenin-POD antibody    
POD Substrate    
Substrate Enhancer    
Washing Buffer 10x concentrated    
Sample Buffer    
Lysis Buffer 5x concentrated    
Microplates (strip frame with 12 modules of 8 wells), precoated with anti-β-Gal (2)    
Self-adhesive Plate Cover Foils (3)    
Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport

Documents

Certificate of Analysis

Protocols & Articles
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