Download Live Cell Luciferase Assay Kit

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Product Information Sheet (0626-005)
Live Cell Luciferase Assay Kit - Product M0626
NOTE: The following information is given as a viable methodology for use of the MGT Live Cell
Luciferase Assay Kit. The user may determine their own best conditions for use dependent on the
specific conditions present in their experiment.
I. OVERVIEW
A variety of species have evolved the ability to emit light biochemically. Among
those that have found the most use in molecular biology applications are the North
American firefly photinus pyralis and the bacterium Vibrio harveyi. The firefly
luciferase gene (luc) has been cloned into a variety of species [Brasier, et al., 1989;
Riggs, et al., 1987; Ow, et al., 1986; Lemonick, et al., 1986; deWet et al., 1987;
Keller, et al., 1987; Nordeen, et al., 1988]. Measurement of transciptional regulation
by coupling the expression of a reporter gene is widely used to monitor a variety of
physiological events including the action of specific receptor elements on gene
expression, signal transduction, expression of transcription factors, protein-protein
interactions, viral infection and processing [Alam, et al., 1990; Wood, K.V., 1991].
The firefly luciferase gene encodes a functional protein immediately upon
transcription, and an assay for the resulting light production is highly-sensitive (very
low background), rapid and reliable, making luciferase a popular choice for reporter
cloning experiments. These assays are also well suited to automation and highthroughput applications.
Firefly luciferase (Photinus-luciferin:oxygen 4-oxireductase[EC1.13.12.7]) is a 61kD
protein (monomer) that catalyzes the mono-oxygenation of the substrate D-luciferin
as shown in Figure 1 below.
HO
N
S
S
N
COOH
ATP Mg+2
HO
N
S
S
N
O
[E]
M0237
+PPi
O2
[E]
HO
N
S
S
N
-CO 2
O-
oxyluciferin
+ light (560 nm)
AMP
HO
N
S
S
N
+AMP
O
O
O
2
Figure 1. Luciferase mechanism for D-luciferin.
In the presence of D-luciferin, O2 and ATP, an enzyme-bound luciferyladenylate
complex is formed, followed by oxidative decarboxylation with the production of
CO2, oxyluciferin, AMP and light. (The luciferase reaction can be, therefore, often
used to monitor ATP concentration.) The chemical energy of the in vitro reaction
can proceed with a high quantum yield (0.88 at pH 7.9).
DETAILS ON PERFORMING THE ASSAY
Firefly luciferase luc assays can be grouped into two general classes based on
whether the luciferase activity is measured in cell lysates or homogenates (Solution
Assay), or in vivo in intact cells or tissues (Cellular Assay). In both these types of
assay conditions, it is recommended that standard or control assays be performed
simultaneously using purified enzyme if possible. The light output from the
luciferase assay can be measured as peak luminescence or as an integrated light
output which includes peak luminescence (typically 1-60 sec. duration). Both of
these values should be proportional to luciferase concentration. The light emission
from the enzyme reaction peaks within seconds at a maximum of 562 nm (green
color) and using the purified enzyme in the presence of excess substrate(s), the light
output is proportional to the concentration of luciferase present.
Recommended Protocol.
The sensitivity of firefly luciferase assay can vary depending on several factors
described in assay conditions below. The optimal luciferase assay conditions will
need to be measured in each experiment to maximize the luminescence signals.
1. Prepare the assay reagent by mixing the D-luciferin reagent with ATP (0626001) with the Reaction Buffer (0626-002). Transferring one bottle (10mL) of
Reaction Buffer into one D-luciferin reagent bottle gives an assay reagent
with final concentration of 1mM D-luciferin and 2mM ATP in solution and
ready to use. (NOTE: Adding a different volume of Reaction Buffer can
change the final concentration of D-luciferin and ATP for different/optimal
assay conditions).
***In the case of live cell assays, instead of dissolving the substrate (Dluciferin) in reaction buffer, as you would for the lysis assay, simply dissolve
the Luciferin and ATP in the appropriate media and incubate. Refer to CELL
CULTURE ASSAY below for more information on live cell staining protocols.
2. For lysis of luc+ recombinant cells, add Cell Lysis Buffer (M0626-003)
(approximately 1 mL of buffer per 100 mm plate of cells (approx. 80%
confluent) grown in culture, and incubate for 10-30 min. Final concentration
approximately 105 to 106 cells/mL.
3. Combine 100-200 µL of assay reagent with cell lysates from the luciferase
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recombinant cells (100 µL), to cells washed with PBS, or with a purified
luciferase enzyme solution as a positive control.
4. Detect luminescence using different methods, such as CCD camera,
luminometer or microplate reader. The luciferase activity reaches peak
intensity quickly (within 10-30 min.), and the immediate measurement of the
luminescence signals is recommended.
SOLUTION ASSAY
One method of assaying the expression of the luc gene in transiently transfected cell
lines is by lysis of the recombinant cells and assay of the supernatant cell
suspension for luciferase activity. Several important factors will govern whether the
results of this assay are optimal.
1. Lysis conditions.
The method of cell lysis may affect subsequent luciferase activity. Freeze-thaw cell
lysis techniques may cause denaturation of the temperature sensitive luciferase
enzyme and reduce its activity. A detergent mediated cell lysis technique,
employing the non-ionic detergent Triton X-100 is found to improve luciferase
activity over freeze-thaw techniques by as much as 25-fold. This is due to both
better stabilization of the enzyme and more thorough extraction of the enzyme. A
detergent mediated cell lysis buffer is provided in this kit. Modified detergent
homogenization or sonication buffer solutions without the chelator EGTA have also
been used. For mammalian tissue samples, homogenize appoximately 5mg of
tissue (wet weight) to isolate 1 x 105 cells in 1.0 mL of lysis buffer, for assay (step 2,
above). Enzyme recovery will be dependent upon tissue type and preparation
conditions.
Extracts are centrifuged (13000 x g, 5-10 min.) and the supernatant solutions are
removed and stored at –70oC. Note that only slightly warmer storage temperatures
(-20oC) cause reduction of luc encoded luciferase activity. Vortex mixing of
supernatant solutions prior to assay is suggested.
An alternative homogenization buffer has been employed for detection of luc
expression in transfected insect larvae containing: PBS (50mM, pH 7.3) with 2 mM
phenylmethylsulfonyl fluoride. The toluenemethylsulfonyl fluoride was presumably
added as a protease inhibitor.
2. Assay Conditions.
Cell lysates of homogenates are typically assayed as described below. A crude
tissue homogentate or cell lysate (50 – 100 µL is suspended in the MGT Reaction
Buffer (usually 1x to 5x the sample volume) at 20oC containing the D-Luciferin
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Reagent with ATP (0.1 to 1.0 mM final concentration) and the light emission
measured by one of the techniques described in section I.
For standardization, control reactions with purified enzyme are recommended,
since the kinetics of the light reaction using a crude enzyme preparation is quite
different from those observed with the purified crystalline luciferase enzyme. The
difference in activity for the crude enzyme is often due to the presence of a number
of contaminating enzymes, the presence of inorganic pyrophosphate or
dehydroluciferin. Several reports indicate that the firefly luciferase enzyme prefers a
lipidic environment for optimal activity. Therefore, partially purified enzyme
preparations sometimes are found to have greater activity. Added cholinecontaining phospholipids like dipalmitoylphosphochloine (DPPC),
dioleoylphosphochloine (DOPC) or dioleoyl-L-serine (DOPS) may increase stability
of the enzyme during and after purification.
For the standard assay, a linear response for light output with respect to luciferase
protein concentration should be obtained, with intercept at the origin. Luciferase
activity is expressed in light units (LU) per µg of protein. As described above, the
light output can be measured as an integral of light emission over a defined time
length (most common) or as the optimum light output (peak). In either case,
standard curves should show a linear relationship.
Of course, additional analyses for luciferase expression (for example, gel
electrophoresis) can be performed in which a standard luciferase band (62kD) is
also run.
CELLULAR ASSAY
An exciting method of measuring luc gene expression involves analysis in intact
cells and tissues. This technique has been widely used in the analysis of luc activity
in transgenic plants [Schnieder, et al., 1990], transgenic fish [Tamiya, et al., 1990],
viruses and bacteria [Jacobs, et al., 1993], mammalian cells and yeast [Hooper, et
al., 1990].
Previously, luciferase expression in mammalian cells was typically measured by
luminometer analysis of cell lysates on addition of substrates and ATP. This
method has the major drawback of destroying the biological sample, thus
preventing further analysis and isolation of luciferase expressing cells. The use of
charge-couples device (CCD) cameras to observe directly intact mammalian cells
expressing luciferase can provide integrated signal output over time, and the digital
images can be used for spatial and quantitative analysis. Microtiter plate assay
systems also utilize this technology in high-throughput format.
A concern in assay of the luc reporter gene in living tissues and cells is whether the
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in vivo pattern of light production is consistent with the expression of luciferase and
other parameters, such as the permeability, stability, and availability of the
substrates, luciferin, molecular oxygen and ATP or the tissue specific distribution of
the cloned luciferase enzyme to individual organelles. Luciferase is not a cytosolic
enzyme, as are many other cloned enzyme activities (lacZ, GUS, etc.) but becomes
localized in peroxisomes in fireflies, plants, yeast and mammalian cells. Transport
of luciferin and other substrates must therefore pass through both the plasma and
peroxisomal membranes prior to reaction. Redirecting specific expression of the
luc gene to other subcellular compartments has been accomplished using various
promoter-luciferase constructs [Schneider, et al., 1990] for transfection, but common
cloning techniques still express luciferase as a peroxisomal enzyme.
In general, attempts to measure luc activity in vivo has utilized chemical means for
delivery into the cells. To optimize transport of luciferin into cells in culture, assay
conditions employing a low pH buffer (100 mM Na-citrate, pH 5.2) cause
protonation of the carboxyl group of D-luciferin, and increase delivery. In addition,
the use of co-solvents (up to 10% DMSO) has also been used to permeabilize the
cell membrane. Luciferin, in these studies, is present in excess, usually at 1 mM
concentration.
CELL CULTURE ASSAY
Cells expressing the luc gene are grown under normal conditions to near
confluency. The medium is removed and replaced with an assay buffer containing:
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•
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Dulbecco’s Modified Eagle medium (DMEM)
Sodium bicarbonate (to pH 6.9)
Dimethylsulfoxide (DMSO, 15%)
1 mM D-Luciferin plus ATP (2mM)
Alternately, the ionophore nigericin can be used to permeabilize cells, and the
sterile buffer containing the following reagents is used:
•
•
•
•
•
DME with sodium bicarbonate (to pH 7.3)
3.5 mM KCl
60 mM NaCl
1 uM nigericin
D-luciferin (1 mM) + ATP (2mM)
Please note that optimal ion concentration for permeabilization and optimum light
output may vary from one cell line to another.
ASSAY IN INTACT TISSUES
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Detection of luc gene activity in tissues and whole organisms requires
permeabilization through several cell layers. Luckily, because of the vasculature of
plant tissue, analysis of luc gene expression in most intact plant tissues simply
involves immersing the plant tissue (intact leaves, leaf disks, petals, root tissues,
etc.) in a reaction solution containing luciferin (0.4 mM) in a Murishage and Skoog
salt medium or the like for up to 1 hour. Alternately, luminescence in leaves is
recorded by soaking transgenic leaves in a buffer solution containing 1 mM luciferin;
2 mM ATP; 100 mM sodium citrate (pH 5.0) and 20 % dimethylsulfoxide (DMSO)
for 2.5 hours.
Introduction of luciferin through roots appears to be the most efficient method of
substrate uptake. Luciferin was introduced through roots into whole plants using a
sterile solution of 1 mM luciferin plus ATP (2 mM) for 2-5 hours. The pattern of
vascular transport of the substrate is examined in whole plants over this time frame.
In addition, once the plant tissue is exposed to a luciferin solution, the light emission
reaction continues unabated for several days, allowing studies into the control of luc
gene expression in vivo.
Contact or normal photography is a common method of measuring the light
emission patterns from these assays. Suggested film types include: Polaroid type
47 (ASA 3000) or 612 (ASA 20,000); Kodak Royal X-pan 4166 or type 103aF film;
or Kodak OG-100 or OG-1 X-ray films.
These materials are intended for research purposes only. Use in drug or
manufacturing processes is strictly prohibited. Please contact us for information on
use or licensing.
MATERIALS
A.) D-Luciferin with ATP Reagent (0626-001). Crystalline powder with ATP
disodium salt. Dilute with 10 mL of Reaction Buffer to provide a 1mM solution of Dluciferin containing 2mM ATP.
B.) Buffer Solutions.
Cell Lysis Buffer (0626-003). 25mM Tris-phosphate (pH 7.8) containing
10% glycerol, 1% Triton X-100, 1 mg/ml BSA, 2 mM EGTA and 2mM DTT.
Reaction Buffer (0626-002). 25mM Glycylglycine, 15mM MgSO4, 4mM
EDTA, 15mM Potassium phosphate pH 7.8, 1mM DTT, and 1mM Coenzyme A.
Luciferase Inhibitor (product M0236) D-Luciferin, 6’-O-methyl ether (not
provided).
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Storage and Handling.
Chemiluminescent reagents and inhibitors should be handled with care, kept cold
(ice-bath) while in use, and stored frozen (below -20°C). In case of contact with skin
or eyes, wash thoroughly with soap and cold-water. Reagents should be stable for
at least 6 months prior to use, following purchase if stored frozen (below -20°C).
Low light readings for blank samples will indicate decomposition. After dissolving
the D-Luciferin Reagent with ATP in reaction buffer or media, store frozen. ATP
decomposes quickly in solution overnight.
References:
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"Transmembrane Motility Assay of Transiently Transfected Cells by
Fluorescent Cell Counting and Luciferase Measurement." J.J. Gildea, et al.
Biotechniques 29: 81 (2000).
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"The Use of the Luciferase Reporter System in Plant Gene Expression
Studies." W. Van Leeuwen, et al. Plant Mol. Biol. Rep. 18:143a (2000).
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"Reporter Genes and Transient Assays for Plants." B.F. Matthews, et al.
Methods Mol. Biol. 55:147 (1995)
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"Chemiluminescent and Bioluminescent Reporter Gene Assays." I.
Bronstein, et al. Anal. Biochem. 219: 169 (1994)
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"Firefly Luciferase, Synthesized to Very High Levels in Caterpillars Infected
with a Recombinant Baculovirus, Can Also Be Used As an Effective
Reporter Enzyme In Vivo." P.K. Jha, et al. FEBS Lett. 274: 23 (1990)
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"Transient and Stable Expression of the Firefly Luciferase Gene in Plant
Cells and Transgenic Plants." D.W. Ow, et al. Science 234: 856 (1986).
M0626 Kit Contents
Description
Quantity
Part No.
Storage
Reagents
D-Luciferin (2.8mg)
with ATP added
Reaction Buffer
1 vial
1 x 10ml vial
0626-001
0626-002
F,L,B_______
C
Cell Lysis Buffer
1 x 10ml vial
0626-003
C
Documentation
MSDS sheets
1
0626-004
N/A
Product Information sheet
1
0626-005
N/A
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o
o
Notes: F=store at or below -20 C; R=store at room temperature; C=store cold (4 C); L=light
sensitive; D=store desiccated FL=flammable; G=wear protective clothing/gloves/safety glasses
when using; B=avoid breathing dust/fumes; R=read protocol instructions carefully prior to use.