study of the lac operon has played an important role
in understanding the control of gene expression in
bacteria. In prokaryotes, gene expression is controlled
primarily at the level of transcription. For eukaryotes,
the promoter activity can be analyzed by using fusion
genes containing the promoter of interest attached
to the bacterial b -galactosidase gene. The level
of b -galactosidase expression indicates the level
of transcription under different regulatory conditions.
provides a sensitive, quantitative assay for b -galactosidase.
Cleavage of 4-methylumbelliferyl-b -D-galactoside
by b -galactosidase yields the fluorescent molecule
4MU). 4-methylumbelliferone is fluorescent above pH
8. When excited by 365nm light, 4MU emits light at
460nm. Used with the Turner BioSystems TD-700 Laboratory
Fluorometer, the assay is sensitive enough to detect
picogram levels of b -galactosidase.
Laboratory Fluorometer with standard PMT and 10mm
x 10mm square cuvette adaptor (P/N 7000-009).
UV lamp (P/N 10-049).
Wavelength UV Filter Kit (P/N 10-302R) which includes
300-400 nm excitation filter (P/N 10-069R) and emission
filter (P/N 10-110R-C).
x 10mm square methacrylate disposable cuvettes (P/N
sodium salt, MW=198.20
carbonate, anhydrous, MW=105.99
mM Tris-HCl, pH 7.5
mM 4-methylumbelliferyl-b -D-galactoside (0.1 mg/ml)
(w/v) trichloroacetic acid
4MU SOLUTION PREPARATION
4MU stock solution A (1mM):
Add distilled water to 100ml
Store at 4░C, away from light.
4MU stock solution B (1ÁM):
10 Ál 4MU stock solution A
Dilute with 10 ml distilled water.
Store at 4░C, away from light.
Carbonate stop buffer (0.20M):
Sodium carbonate, anhydrous, MW = 105.99
Add distilled water to 1000ml.
Glycine-Carbonate stop buffer
pH to 10.7
4-Methylumbelliferone standard, 50nM:
Ál 1 uM 4MU solution
1.9 ml Glycine-Carbonate stop buffer
Prepare just before use.
mM Tris-HCl, pH to 7.5
125 mM NaCl
2 mM MgCl2
12 mM 2-mercaptoethanol
0.3 mM 4-methylumbelliferyl-b -D-galactoside (0.1
mg/ml) substrate. Prepare reaction cocktail minus
substrate at room temperature. Disperse substrate
into a small volume of ethanol (0.5% of the total
volume of cocktail). Add ethanol- substrate solution
to acqueous cocktail. Then rapidly vortex until
(w/v) trichloracetic acid
Accurate pipetting and thorough mixing are critical
for reproducible results. However, take extreme care
when mixing samples; do not introduce air bubbles.
Air bubbles can cause scattering of light leading
to inaccurate results. If air bubbles form, hold the
upper portion of the cuvette in one hand and gently
tap the bottom sides of the cuvette with your other
hand to release bubbles.
Install filter 10-069R excitation and emission filter
10-110R-C. For further assistance refer to Section
III (Optical Filter Installation and Removal) on page
9 of the TD-700 Operating Manual.
Insert the Near U.V. Mercury Vapor lamp (P/N 10-049).
Then turn on the instrument and allow a 10 minute
warm-upperiod. For further assistance refer
to Section IV (Lamp Installation and Removal) on page
11 of the TD-700 Operating Manual.
Select units of measurement. Press <MENU>, <2>,
and select desired units.
Prepare 2ml volumes of 50, 100, 150, and 200 nM 4MU
standards in Glycine-Carbonate stop buffer.
Calibrate the instrument with standard prepared from
step 4.2. Mix well. Insert cuvette containing standard
into the instrument, close the lid, and press <CAL>.
Enter in the actual concentration of the standard
or a convenient value that will display a multiple
of the actual DNA concentration, then <ENT>.
If a value other than the actual concentration is
used, be sure to note the multiplication factor used.
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.
Blank the instrument with Glycine-Carbonate stop buffer.
Place 1.9 ml assay solution into a cuvette, insert
into the instrument, close the lid, and press <BLANK>.
Press <1> to accept blank value. Remove cuvette.
Add 40 Ál of sample to a centrifuge tube. Use water
for a blank.
Add 160 Ál of the reaction cocktail.
Incubate at 370C for 30 minutes.
Stop the reaction by adding 50 Ál of 25% TCA. Cool
Clarify the solution by centrifugation in a microcentrifuge
for 1 to 2 minutes.
Add 100 Ál of supernatant to 1.9 ml of Glycine-Carbonate
Read the fluorescence by inserting the cuvette into
the fluorometer, closing the lid, and pressing <GO>.
Determine the concentration from the standard curve
in section 4.
A 40 Ál sample should contain 10-6 to 10-5
units (nM 4MU min-1) of b -galactosidase
activity. If other dilutions of the sample into the
reaction cocktail are used, adjust the samples accordingly.
The Glycine-Carbonate reagent is sufficient to titrate
1/3 its own volume in 5% TCA to the proper pH for
reading (200 Ál reaction stopped with 50 Ál of 25%
The protocol is designed for assay of E.coli b -galactosidase
activity, which is active at neutral pH. The vertebrate
form of b -galactosidase is a lysosomal enzyme, which
has optimal acitivity at pH 4.5. The lysosomal activity
can be assayed in acetate buffer instead of Tris buffer.
The assay should be performed at both pH values when
lysosomal contamination of reactions is suspected.
Depending on the calibration setting used, maximum
readings are obtained at 40 to 200nM 4MU final concentration
in the Glycine-Carbonate buffer.
Limitations on the sensitivity of the assay are determined
by the background fluorescence of the substrate. It
is therefore important to use freshly prepared substrate
solutions in assays where high sensitivity is desired.
Frozen reaction cocktails may be used, but backgrounds
gradually increase with repeated freeze/thaw cycles.
In addition, substrate sometimes precipitates from
frozen cocktails. Resolubilization is slow unless
the cocktail is heated to 37░C and repeatedly
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