TBS-380 Mini-Fluorometer Method for
The gene for ß-galactosidase
is commercially available in a variety of configurations for reporter gene
studies. The recombinant ß-galactosidase enzyme can be routinely manipulated
and assayed to study promoter function, tissue specific expression,
developmental regulation, mRNA stability, and signal sequences that target
proteins for various organelles. The advantages of using ß-galactosidase to
report the activity of promoters and genes are two-fold; assays are
straightforward, and substrates for enzymatic analysis are readily available. ß-galactosidase
activity in solution can be revealed by the fluorogenic substrate
4-methylumbelliferone in a sensitive, quantitative assay using the TBS-380
2. HOW IT
4-methylumbelliferone (4-MU) do not fluoresce unless cleaved to release the
fluorophore. Fluorometric enzyme assays are based on the hydrolysis of
4-MU-containing substrates such as ß-4-MU-glucuronide by
ß-glucuronidase(GUS), or ß-4-MU-galactose by ß-galactosidase (GAL). Cleavage
of 4-methylumbelliferyl-ß-D-galactoside by ß-galactosidase enzyme yields the
fluorescent molecule 4-MU that emits light at 460 nm when excited by 365 nm
BioSystems TBS-380 Mini-Fluorometer provides sensitive, reliable measurements of
4-MU. Using standard 10x10 mm cuvettes, the TBS-380 has a linear detection range
from 750 nanomolar down to 0.1 nanomolar, or
20 fg/mL 4-MU. Sensitivity levels for the minicell adaptor range from 200
nanomolar down to 1.0 nanomolar.
coefficient of variance (CV) for three replicates of 15 dilution points was
3.5%. The variability seen in replicate measures is associated with
photo-bleaching and inherent fluctuations in 4-MU assays. This result includes
measurements from minicell and 10x10 mm cuvettes.
4-MU detection using the TBS-380 Mini-Fluorometer.
Replicate fluorescence measures of 4-MU serial dilutions were averaged and
plotted. Two overlapping curves were generated using two different calibration
values, 500 nM and 50 nM, for greater accuracy. R-square values are shown for
both curves and indicate linear relationship between fluorescence and 4-MU
concentration. The Minimum Detection Limit (MDL) for 4-MU using the TBS-380
Mini-Fluorometer is 0.1 nM using a 99.5% confidence limit
Mini-Fluorometer with UV optical configuration (P/N 3800-003)
• 10x10 mm Methacrylate Fluorescence Cuvettes (P/N 7000-959)
• Minicell Adaptor Kit, optional, (P/N 3800-928)
• Minicell Borosilicate Cuvettes, optional, (P/N 7000-950)
• 4-methylumbelliferone, sodium salt, MW=198.20 (Sigma, P/N 1508)
• Distilled water
• Sodium carbonate, anhydrous, MW=105.99
stock solution A (1mM):
19.8 mg 4-methylumbelliferone (sodium salt), MW = 198.20
Add distilled water to 100 mL
Store at 40C, away from light.
4.2 4-MU stock solution B (1mM):
10 mL 4-MU stock solution A
Add 10 mL Distilled water
Store at 40C, away from light.
4.3 Carbonate stop buffer (0.20 M):
2.12 g Sodium carbonate, anhydrous, MW = 105.99
Add DIH2O to 100 mL.
In order to
measure 4-MU for reporter gene assays, the ß-Gal producing cells needs to be
lysed and incubated with the appropriate substrate. Commercial kits using ß-Gal
reporter genes typically include treatment protocols and signal enhancers
specific to tissue and recombinant enzyme. These application notes are based on E.
coli ß-galactosidase activity that is active at neutral pH. However, the
vertebrate form of ß-galactosidase is a lysosomal enzyme, which has optimal
activity at pH 4.5 in acetate buffer. Buffer conditions during incubation should
not affect the TBS-380's sensitivity to detect 4-MU fluorescence.
cells and incubate with 4-MU containing substrate according to reagent
manufacturer directions. Incubate all samples for the same period of time,
generally 2 minutes.
5.2 Add 100 µL of cell lysis incubation to 1.9 mL Carbonate Stop
buffer to stop ß-Gal enzyme activity and prepare sample for measurement.
A STANDARD CURVE
Free 4-MU can be
used as a standard to calibrate ß-galactosidase activity in cell cultures or
Generating a standard curve verifies the linearity of the assay within a
particular concentration range. It is recommended that you perform this at least
once when working with a new instrument or performing the assay for the first
time. Also, you may want to generate a standard curve every few weeks as a
quality check on the standard, a reliability check on the instrument, and a
consistency check on technique.
sure the TBS-380 is set to UV optical configuration. If you are using the
minicell adaptor, make sure it is placed in the chamber with "UV"
indicator visible from front.
6.2 Prepare blank by adding Carbonate Stop Buffer to the 10x10 mm
cuvette or minicell cuvette.
6.3 Add 100 µL stock solution B to 1.9 mL Carbonate Stop Buffer (final
conc. 50 nM). Add 5.0 µL solution B to 45 µL Carbonate Stop Buffer when
using the minicell.
6.4 Set the standard value to 50 by pressing [STD VAL]. Use the arrow
keys to raise and lower the values.
6.5 Press [CAL] to calibrate. Press [ENTER] to continue. Insert the
blank and measure as directed. Insert the dilution and measure. Press [ENTER]
to accept calibration values.
6.6 Read remaining samples in dilution series.
(2 mL total)
(100 mL total)
Calibrate the instrument with a dilution near the average fluorescence of your
samples, typically 100 nM to 500 nM (steps 5.3 through 5.5).
7.2 Measure unknown sample by transferring
2 mL from step 4.2 into 10x10 mm cuvette, or transfer 100 µL into minicell
cuvette. Insert cuvette and press [READ].
7.3 Record results or use spreadsheet interface to import data into an
Ziebold, T. O.
(1967) Anal. Chem. 39, 858.