b-Galactosidase or b-D-Galac-tosidase galactohydrolase enzyme has been detected in numerous microorganisms, animals and plants. In some E. coli strains about 5% of the total protein content is b-Galactosidase if lactose is the sole source of carbon. Ist large size makes it less suitable for EIH but is one of the most commonly used enzymes for reporter gene assays. bGal -Juice can used for a wide range of applications in cell based assays were the Gene for b-Galactosidase (lacZ) is used as reporter system.
PJK´s series of stabilized chemiluminescent 1,2-dioxetanes substrates can detect ß-Galactosidase enzyme at low levels:
- Reporter Gene Assays
- Yeast Two-Hybrid
Store the reagent bottles at 4-8 °C or at -20 °C.
Equilibrate the reagent at room temperature till clear solution observed if stored at -20 °C.
Do not contaminate the β-Galactosidase substrate with β-Galactosidase enzyme or other proteins.
β-Galactosidase substrate has a wide range to detect β-Galactosidase enzyme in solution as well as on membrane. The lower enzyme concentration may be atto grams and the highest concentration of β-Galactosidase enzyme may be nano gram depending on the source of the β-Galactosidase enzyme.
For good results, wash your tube or micro titer plate or membrane with 0.1M phosphate buffer, pH 7.2 to 7.4, and then use the substrate.
Best results for chemiluminescence detection of β-Galactosidase enzyme or reporter assays can be obtained from 30 minutes to 45 minutes incubation of β-Galactosidase substrate with β-Galactosidase enzyme and read the plate or tube within 2 to 10 minutes after triggering the reaction mixture by accelerator or enhancer.
Preparation of Cell Lysates for measurement of b-Galactosidase:
For cell lysis, we suggest the follow buffers:
- Animal cells: 100 mM potassium phosphate (pH 7,8), 0,2% Triton X100
- Yeast cells : 0,5 M sodium phosphate (pH 7,1) 50 mM KCl, 5 mM MgSO4
Standard Protocol for Detection of β-Galactosidase in Microtiter Plate Luminometer
Equilibrate at room temperature for 30 minutes before use.
Best results for chemiluminescence detection of β-Galactosidase enzyme or reporter assays can be obtained from 30 minutes to 60 minutes incubation of β-Galactosidase substrate with β-Galactosidase enzyme and read the plate or tube within 2 to 10 minutes after triggering the reaction mixture by accelerator or enhancer.
- Add 5 to 10 μl of diluted β-Galactosidase enzyme or cell extract to microplate wells.
- Add 50 to 100 μl of diluted β-Galactosidase substrate and incubate for 30 to 60 minutes at room temperature.
- Add 50 to 100 μl of triggering reagent and shake it for 10 seconds.
- Place the microtiter plate in luminometer and start reading.
- Best results are obtained between 2 to 10 minutes.