FluoReporter® lacZ/Galactosidase Quantitation KitMP 02905
Revised: 22March2001
Product Information
FluoReporter® IacZ/Galactosidase Quantitation Kit (F-2905)
Introduction
The E. coli lacZ gene, encoding β-D-galactosidase, is exten-
sively used as a reporter gene for detecting the expression of
recombinant fusion genes and for monitoring transfection effi-
ciency in mammalian, yeast and bacterial cells. Although mam-
malian cells do contain β-galactosidases, they are generally
lysosomal enzymes with low pH optima and therefore exhibit
low activity at neutral pH. Combining this with the fact that E.
coli β-galactosidase has a high turnover rate, the enzyme can be
detected at very low levels, making it a sensitive reporter of gene
expression.
The FluoReporter
®
lacZ/Galactosidase Quantitation Kit
(F-2905) contains the improved fluorogenic substrate CUG for
the sensitive detection of β-galactosidase activity. The advan-
tages of this substrate over the commonly used 4-methylumbelli-
feryl β-
D-galactopyranoside (MUG) substrate include its higher
aqueous solubility and increased fluorescence efficiency, resulting
in a lower threshold of detection. Molecular Probes scientists
have demonstrated a practical detection limit of 0.5 picograms of
β-galactosidase using this FluoReporter kit and a fluorescence
microplate reader. Although this kit was originally developed for
assaying mammalian cell extracts, it is very useful for enzyme-
linked immunosorbent assays (ELISAs)
1
that employ β-galacto-
sidase conjugates, as well as for assaying bacterial
2
and yeast
cell extracts, prepared using conventional protocols.
3-6
Each kit
supplies sufficient reagents for at least 1000 tests using 96-well
microplates.
Materials
Reagents Supplied
• Substrate reagent (Component A), 3 mL of 40 mM
3-carboxy-umbelliferyl β-
D-galactopyranoside (CUG) in
100 mM sodium phosphate buffer (pH 7.0), 1 mM MgCl
2
and
110 mM β-mercaptoethanol
• Reference standard (Component B), 1 mL of 10 mM
7-hydroxycoumarin-3-carboxylic acid in dimethylformamide
Storage and Handling
The CUG substrate reagent should be stored frozen at -20°C;
the storage temperature for the reference standard is not critical.
Both components should be protected from light. When stored
properly, these solutions are stable for at least six months. CUG
solutions should not be exposed to room temperature for ex-
tended periods of time as spontaneous hydrolysis will occur. If
the CUG substrate exhibits a high fluorescent background signal
prior to enzymatic hydrolysis, then this reagent may have deterio-
rated during storage and is likely no longer suitable for use.
Materials Required but Not Provided
• Reaction buffer
• Stop buffer
•β-galactosidase (for calibration curve, if desired)
Protocol
Overview
The FluoReporter lacZ/Galactosidase Quantitation Kit pro-
vides a sensitive method for measuring the level of β-galacto-
sidase activity in solution or in cell extracts. Enzymatic cleavage
of the fluorogenic β-galactosidase substrate CUG yields 7-hy-
droxycoumarin-3-carboxylic acid, a highly fluorescent product
that can be quantitated in a fluorescence microplate reader.
The CUG substrate and an aliquot of cell extract or other
β-galactosidase*containing solution are added to each micro-
plate well. After an incubation period, the reaction is terminated
by adding stop buffer, which raises the pH of the sample. The
fluorescence of each sample is then measured using a fluores-
cence microplate reader. The fluorescence signal can be con-
verted into picograms of β-galactosidase by using a standard
curve, if desired. This assay has a linear detection range of about
0.5 to over 1000 pg β-galactosidase (Figure 1). The lower detec-
tion limit corresponds to about ten lacZ-positive NIH 3T3 cells
per well. The following protocol describes the assay of mamma-
lian cell extracts using a fluorescence microplate reader and may
require some modification when assaying bacterial or yeast cell
extracts
1-4
or β-galactosidase conjugates in an ELISA.
5
Solution Preparation
1.1 Prepare a reaction buffer containing 0.1 M sodium phos-
phate, pH 7.3, 1 mM MgCl
2
and 45 mM β-mercaptoethanol.
Approximately 10 mL of reaction buffer are needed per 96-well
microplate. Additional buffer is needed for preparing enzyme
dilution buffer, which is used when generating a standard curve.
1.2 If generating a standard curve, prepare enzyme dilution
buffer by adding bovine serum albumin (BSA) at a final concen-
tration of 1 mg/mL to approximately 1 mL of reaction buffer.
Storage upon receipt:
20°C
Protect from light
Ex/Em of reaction product: 386/448 nm
FluoReporter® lacZ/Galactosidase Quantitation Kit2
1.3 To generate a standard curve, prepare a fresh 1 µg/mL solu-
tion of β-galactosidase solution in enzyme dilution buffer. In
addition, make 10-fold, 100-fold, 1000-fold and 10,000-fold
dilutions of this enzyme solution in enzyme dilution buffer
(note A). These solutions are stable for several hours at room
temperature. In the enzyme assay, 10 µL of each of these solu-
tions will be pipetted into an individual microplate well, yielding
10ng, 1 ng, 100 pg, 10 pg and 1 pg β-galactosidase standards,
respectively. For increased accuracy, we recommend performing
assays in triplicate.
1.4 Prepare a 1.1 mM working solution of the CUG substrate
reagent by diluting 275 µL of the 40 mM stock solution (Compo-
nent A) with 9.73 mL of reaction buffer. CUG solutions should
not be exposed to room temperature for extended periods of time
as spontaneous hydrolysis will occur. Approximately 10mL of
CUG working solution are needed for a 96-well microplate. The
working solution can be stored frozen at -20°C for at least six
months.
1.5 If desired, prepare a 0.1 mM working solution of the
7-hydroxycoumarin-3-carboxylic acid reference standard by
diluting the stock solution (Component B) 100-fold with reaction
buffer. The reference standard can be used to normalize fluores-
cence, allowing assays performed at different times, or on differ-
ent instruments, to be compared to a single standard curve.
1.6 Prepare a stop buffer containing 0.2 M Na
2
CO
3
. Approxi-
mately 5 mL of stop buffer are needed for a 96-well microplate.
Enzyme Assay
2.1 Carefully pipet 10 µL cell extract into individual microplate
wells (note B). It may be necessary to dilute the cell extracts
prior to performing this assay, depending on the level of β-galac-
tosidase present. For more accurate results, we recommend per-
forming each assay in triplicate.
2.2 If generating a standard curve, pipet 10 µL of each of the
purified β-galactosidase solutions prepared in step 1.3 into indi-
vidual microplate wells.
2.3 Pipet 10 µL of reaction buffer into a microplate well to serve
as a blank.
2.4 Add 100 µL of the CUG working solution (prepared in
step 1.4) to each well.
2.5 If desired, pipet 100 µL of the 0.1 mM reference standard
(prepared in step 1.5) into an empty well. The reference standard
can serve as an instrument-independent control. Normalization
of the fluorescence signals with the reference standard allows a
single standard curve to be used for assays performed at different
times, even if performed on different instruments or with differ-
ent instrument settings. The reference standard can also be used
to convert the fluorescence signal into moles of product.
2.6 Incubate the microplate for 30 minutes at room temperature.
For comparison to a previously generated standard curve, incuba-
tion time is critical the same incubation time and temperature
should be used to ensure accurate quantitation.
2.7 Add 50 µL of stop buffer (prepared in step 1.6) to each well.
In addition to terminating the reaction, the stop buffer causes an
increase in the fluorescence of the product.
2.8 Measure the fluorescence of the solution in each well using a
suitable fluorescence microplate reader fitted with an excitation
filter centered at about 390 nm and an emission filter centered at
about 460 nm (notes C, D).
Analysis of Results
3.1 To generate a standard curve, first subtract the fluorescence
of the blank from that of each of the samples containing the puri-
fied β-galactosidase solutions. If the standard curve will be used
for comparison with assays performed at a later date, divide the
background-subtracted fluorescence of the β-galactosidase stan-
dards by the background-subtracted fluorescence of the reference
standard. Plot the resulting corrected fluorescence intensities
versus enzyme amount on a loglog scale. Note that the values
for enzyme amount must be adjusted to compensate for the purity
of the enzyme preparation. Alternatively, fluorescence can be
plotted versus units of β-galactosidase activity. A standard curve
(without reference standard normalization) should resemble the
one shown in Figure 1.
3.2 Analyze the fluorescence of the samples by subtracting the
fluorescence of the blank from that of each sample. If a reference
standard is being used, divide the corrected fluorescence by the
background-subtracted fluorescence of the reference standard.
dase in each well.
Notes
[A] Additional intermediate dilutions (e.g. 3-, 30-, 300- and
3000-fold dilutions) can be made to create a more complete stan-
dard curve, if desired.
[B] Cell extracts from mammalian cells can be prepared
using a freezethaw cell extraction protocol or by treatment of
the cells with a detergent, such as Triton
®
X-100. Alternatively,
Figure 1. Example of a standard curve using the FluoReporter
®
lacZ/
Galactosidase Quantitation Kit.
FluoReporter® lacZ/Galactosidase Quantitation Kit3
Product List Current prices may be obtained from our Web site or from our Customer Service Department.
Cat # Product Name Unit Size
F-2905 FluoReporter
®
lacZ/Galactosidase Quantitation Kit *1000 assays* ......................................................................................... 1 kit
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Copyright 2001, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice.
Triton X-100 can be added to the reaction buffer and the assay
procedure altered to allow for cell lysis within the microplate.
For instance, 10 µL of a cell suspension can be added to 90 µL
of reaction buffer prepared with 0.1% Triton X-100 (v/v) and the
microplate incubated at room temperature for 10 minutes to al-
low for cell lysis. The enzyme reaction can then be initiated by
the addition of 10 µL an 11 mM solution of CUG (which is
10-fold higher than the concentration of the working solution
prepared in step 1.4). Bacterial and yeast cell extracts prepared
using conventional methods
1-4
can also be assayed with the kit.
[C] These excitation and emission wavelengths may not corre-
spond to standard filter sets on all microplate readers. Other fil-
ters can be used (e.g. excitation at 360 nm), but sensitivity will
be diminished.
[D] The fluorescence should be measured within 15 minutes of
adding the stop buffer. If comparing samples to a previously
generated standard curve, the same time interval between stop-
ping the reaction and reading the fluorescence should be used.
References
1. Anal Biochem 118, 102 (1981); 2. Infect Immun 61, 5231 (1993); 3. Short Protocols in Molecular Biology, Second Edition, F.M. Ausubel et al., Eds., Greene
Publishing Associates and John Wiley & Sons, New York (1992) pp. 1327; 4. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) p. 16.66; 5. Methods Enzymol 101, 181 (1983); 6. Experi-
ments in Molecular Genetics, J.H. Miller, Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1972) p. 352.
