2012 Promega catalogue

Cell Signaling
cAMP-Glo Max Assay
Product Size Cat.# Price ($)
cAMP-Glo Max Assay 2 plates V1681 517.00
For Research Use Only. Not for Use in Diagnostic Procedures.
20 plates V1682 Pls. Enq.
10 × 20 plates V1683 Pls. Enq.
Description: The cAMP-Glo Max Assay is a homogeneous, bioluminescent
and high-throughput assay to measure cyclic AMP (cAMP) levels in cells.
Compounds that modulate GPCRs coupled with adenylate cyclase typically alter
intracellular cAMP levels. The cAMP-Glo Max Assay monitors cAMP levels
in cells in response to the effect of agonists, antagonists or test compounds on
G protein-coupled receptors (GPCRs). The assay is based on the principle that
cyclic AMP (cAMP) stimulates protein kinase A (PKA) holoenzyme activity,
decreasing available ATP and leading to decreased light production in a
coupled luciferase reaction.
This improved version combines the lysis and cAMP reaction buffers into the
cAMP-Glo ONE Buffer. This new format streamlines the protocol and reduces
the time needed to complete the assay. The new ONE Buffer is supplied at a
5X concentration, which provides increased flexibility for starting cell culture
volumes.
The cAMP-Glo Max Assay can be performed in 96-, 384- or 1536-well
plates. The cells are induced with a test compound for an appropriate period
of time to modulate cAMP levels. After induction, cells are lysed, and the cAMP
released stimulates protein kinase A in the reagent (Figure 1). The Kinase-Glo ®
Reagent is then added to terminate the PKA reaction and detect the remaining
ATP using luciferase reaction. Luminescence is recorded using a plate-reading
luminometer. The half-life for the luminescent signal is greater than four hours,
allowing ample time to read the plates and eliminates the need for luminometers
with reagent injectors.
Features:
Fast and Easy to Use:
• Improved—Lysis and cAMP detection steps combined
(cAMP-Glo ONE Buffer).
• ONE Buffer—5X concentration provides better flexibility for
starting cell culture volumes.
• Assay can be completed in approximately 30 minutes.
Excellent Signal-to-Noise Ratios:
• Best signal:background ratio of all the cAMP assays.
• Signal:Background >200 (with cAMP), >15 (on cells).
• Easily scalable to 1536-well plate formats and beyond.
Proven Luminescent Technology:
• Powered by Ultra-Glo Recombinant Luciferase.
• No interference by fluorescent compounds.
• Nonradioactive.
Storage Conditions: Store the system at –20°C. Before use, completely
thaw all components at room temperature, except for the Protein Kinase
A, which should be kept on ice when not at –20°C. After thawing, mix all
components thoroughly before use. Once prepared, the cAMP detection
solution (cAMP-Glo ONE Buffer with Protein Kinase A) should not be frozen.
Once prepared, the Kinase-Glo ® Reagent should be dispensed into aliquots
and stored at –20°C. See the product label for the expiration date.
Protocol Part#
cAMP-Glo Max Assay Technical Manual TM347
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GloSensor cAMP Assay
Product Size Cat.# Price ($)
GloSensor cAMP HEK293 Cell Line 2 vials E1261 Pls. Enq.
pGloSensor-22F cAMP Plasmid 20 µg E2301 Pls. Enq.
pGloSensor-20F cAMP Plasmid 20 µg E1171 Pls. Enq.
GloSensor cAMP Reagent 25 mg E1290 Pls. Enq.
For Research Use Only. Not for Use in Diagnostic Procedures.
250 mg E1291 Pls. Enq.
Description: The GloSensor cAMP Assay presents a novel approach to
measuring cAMP levels in live cells. cAMP is a key second messenger involved
in signal transduction of GPCRs acting through Gα s and Gα i proteins. The new
assay is based on the GloSensor Technology, a genetically modified form of
firefly luciferase into which a cAMP-binding protein moiety has been inserted.
Upon binding of cAMP, conformational change is induced leading to increased
light output. This live-cell assay excels at kinetic and modulation studies of
signaling through cAMP.
Researchers can use the GloSensor cAMP Assay by transiently expressing
a receptor of interest and the biosensor in the cell line of choice. Alternatively,
stably transfected cell lines with both the biosensor and the receptor of interest
can be made. The protocol is simple: Cells are pre-equilibrated with
GloSensor cAMP Reagent for approximately 2 hours; then cells are treated
with specific agonists/antagonists or compounds, and luminescence is
measured after 10–30 minutes. No other reagent additions or manipulations
are required. Most any common luminometer with injectors is sufficient to read
the assay. GloSensor cAMP Reagent is required for use with this assay per
the GloSensor Limited Use Label License.
Choosing the Appropriate Plasmid:
We offer two variants of the biosensor, and we recommend the pGloSensor-
22F cAMP Plasmid as the first choice for most applications.
pGloSensor-22F cAMP Plasmid. Following cell-free expression in vitro,
the version encoded by this construct shows an increased EC 50 for activation
together with increased signal-to-background ratio at cAMP saturation relative
to the version encoded by the pGloSensor-20F cAMP construct. In general,
we have observed similar relationships between the two constructs when their
performance is compared in living cells.
pGloSensor-20F cAMP Plasmid. The version encoded by this construct
performs well in HEK293 cells at 37°C. Luminescence from the pGloSensor-
22F cAMP Plasmid construct can be more difficult to detect at physiologic
temperatures.
For a more thorough explanation of the general performance differences
between the two plasmids, please consult Section 3.B, Recommendations on
Choice of GloSensor Plasmid, in the Technical Manual (#TM076).
Features:
• Best-in-Class Performance: High Z´ and large signal:background ratio
values. Ideally suited to HTS/uHTS. Up to 1,000-fold changes in light
output obtained.
• Live-Cell, Non-Lytic Assay Format: “Zero-step assay” greatly facilitates
HTS/uHTS. Easy monitoring of cAMP in live cells enables a more complete
analysis of receptor biology.
• High Sensitivity and Increased Biological Relevance: Easy detection
of low-abundance, endogenous receptors; direct detection of G i-coupled
receptor activation and inverse agonist activity in the absence of added
forskolin. PDE inhibitors not needed.
Storage Conditions: Store the pGloSensor cAMP Plasmid at –20°C and
the GloSensor cAMP Reagent at –70°C. Store the resuspended
GloSensor cAMP Reagent at –70°C in single-use aliquots.
Protocol Part#
GloSensor cAMP Assay Technical Manual TM076
49
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Cell Signaling
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