Lumi-Gal 530Lumi-Gal® 530 is a ready-to-use formulation containing Lumigen GPD (4-methoxy-4-(3-β-D-galactosidephenyl)spiro[1,2-dioxetane-3,2'-adamantane]) for the one-step chemiluminescent detection of β-galactosidase. The solution contains the dioxetane, MgCl2, cetyltrimethylammonium bromide and a fluorescent enhancer in 2-amino-2-methyl-1-propanol buffer (pH 9.6). A proprietary enhancer and stabilizing agent promote the efficient operation of β-galactosidase at this pH. This formulation is particularly useful for the chemiluminescent detection of β-galactosidase conjugates in solution.
Addition of Lumi-Gal 530 to a tube, bead or microtiter well at 37°C containing the β-galactosidase results in chemiluminescence which increases with time and reaches a maximum after about 30 minutes. With low concentrations of enzyme, the light intensity remains constant for more than an hour. The luminescence intensity at any time point is a direct measure of the concentration of enzyme.
Principles:Reaction of the chemiluminescent substrate Lumigen GPD with β-galactosidase causes cleavage of the galactoside unit resulting in a moderately stable intermediate. The decomposition of this intermediate in alkaline solution generates chemiluminescence which decays with a half-life of 10 minutes at 37°C in the Lumi-Gal formulation. The combination of the enzymatic hydrolysis followed by the decomposition of the intermediate produces the observed decay curve.
Procedure for testing the reagent:Allow unopened bottle of Lumi-Gal 530 to equilibrate at room temperature with occasional shaking for one hour. Pour enough reagent for one day's use into another polyethylene or polypropylene bottle or, ideally, aliquot the entire bottle into separate smaller bottles. Re-freeze unused portion.
Add 0.1 mL of Lumi-Gal 530 to 8x50 mm or similar tubes. Thermostat tubes at 37°C for 10 minutes.
Prepare, by serial dilution, solutions of purified β-galactosidase or conjugate (e.g. streptavidin-β-galactosidase) in suitable buffer or simply in 2% aqueous BSA. A 1 mg/mL stock solution contains 10-11 to 10-12 moles of enzyme in 5 µL. Prepare dilutions down to 1:1,000,000.
Add 5 µL of enzyme dilution to each tube except blanks. Light emission begins immediately and increases to a maximum over several minutes and then slowly decays. The lifetime of light emission at 37°C ranges from about two hours for the highest enzyme concentration to several hours at lower levels of enzyme.
Measure the light intensity in a suitable luminometer at 20 min or at the maximum for blank and each sample. Light intensity should be linear over the range shown in the accompanying plot.
Lumi-Gal 530 has been carefully formulated to optimize enzyme activity, chemiluminescence intensity and lifetime. It is recommended that for the best performance Lumi-Gal 530 not be diluted by more than 10% through addition of enzyme or other reagents.
Lumi-Gal 530 offers sensitive detection of β-galactosidase conjugates on nylon membranes. Saturate the membrane with Lumi-Gal 530, incubate at 37°C for 30-60 min, drain the membrane and record an image with X-ray film. Multiple exposures may be made since light emission persists for many hours.