2013 Promega catalogue

Cell Signaling
GPCR Assays
cAMP-Glo Assay
Product Size Cat.# Price ($)
cAMP-Glo Assay 300 assays V1501 523
For Research Use Only. Not for Use in Diagnostic Procedures.
3,000 assays V1502 Pls. Enq.
30,000 assays V1503 Pls. Enq.
Description: The cAMP-Glo Assay is a homogeneous, bioluminescent and
high-throughput assay for measuring cAMP levels in cells. The cAMP-Glo
Assay monitors cAMP production in cells in response to the effects of test
compounds on G protein-coupled receptors (GPCR). GPCRs that couple with
adenylate cyclase will increase or decrease intracellular cAMP. 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.
The cAMP-Glo 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 to release cAMP, then
the cAMP detection solution, which contains protein kinase A, is added. The
Kinase-Glo ® Reagent is then added to terminate the PKA reaction and detect
the remaining ATP via a luciferase reaction. Plates are read using a microplatereading
luminometer. Luminescence can be correlated to the cAMP concentrations
by using a cAMP standard curve. The half-life for the luminescent signal
is greater than 4 hours. This extended signal half-life eliminates the need for
luminometers with reagent injectors and allows batch-mode processing of
multiple plates.
Features:
• Fast and Easy to Use: Assay can be completed in approximately
45 minutes. Homogeneous. Two steps following lysis of cells.
• 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. Non-radioactive.
Storage Conditions: Store the system at –20°C. Once prepared, the cAMP
detection solution (cAMP-Glo Reaction 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
cAMP-Glo Assay Technical Bulletin
G protein-coupled
receptor
β
γ
GDP
β α
γ
G protein
Ligand
Inactive
adenylate
cyclase
cAMP
Active
adenylate
cyclase
α
GTP
ATP
Part#
TB357
cAMP-Glo Max Assay
Product Size Cat.# Price ($)
cAMP-Glo Max Assay 2 plates V1681 523
20 plates V1682 Pls. Enq.
10 × 20 plates V1683 Pls. Enq.
For Research Use Only. Not for Use in Diagnostic Procedures.
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 via a luciferase reaction. Plates are read using a microplate-reading
luminometer. The half-life for the luminescent signal is greater than 4 hours
allowing ample time to read the plates and eliminating 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.
Non-radioactive.
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
cAMP-Glo Max Assay Technical Manual
Part#
TM347
7
Drug Discovery
Phosphorylated
protein kinase A
substrate
ADP
P
cAMP
R
R
cAMP
R
R
C C
ATP
Active protein
luciferin
kinase A
+ O 2
Protein kinase A
substrate
luciferase
C C
Inactive protein
kinase A holoenzyme
oxyluciferin
+ AMP
+
Light
6127MA
Section
Contents
Schematic of cAMP production in cells and the cAMP-Glo Assay.
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