AlphaScreen to Measure cAMP Induction with

ALPHASCREEN™ to Measure cAMP induction with SIGNALSCREEN™
Dopamine D1 receptor membranes
Roger Bossé, Lucille Beaudet, Martin Boissonneault, Nathalie Bouchard, Betty Howard**, Chantal Illy, Liliana Pedro,
Geneviève Pinard, Philippe Roby, CJ Wang* & Daniel Chelsky. BioSignal Packard *Packard Bioscience Meriden Connecticut, **Packard Bioscience Downers Grove
AlphaScreen is a novel, homogeneous, nonradioactive assay technology
applicable to a broad range of HTS and assay development applications.
Based on the proximity of two very small beads and an amplified
luminescence signal, AlphaScreen results in a very intense signal output
with robust signal/background ratios. These unique characteristics allow
the technology to be applied to small volume assays without changing
assay component concentrations. Off the shelf beads are available with
a large variety of coatings to make a wide range of assay types possible.
Examples of the assay types currently validated include serine/threonine
kinases, tyrosine kinases, proteases, DNA helicase, functional cAMP,
and a variety of ligand/receptor binding, protein protein interactions,
transcription factor/DNA, and low affinity binding (1 uM) interactions.
The AlphaQuest HTS Microplate Analyzer with a 40-plate stacker and
internal bar code reader allows for rapid processing of assay plates
regardless of sample density, 96, 384 or 1536. In this poster we will
demonstrate the ability to perform cAMP induction or inhibition studies
with SignalScreen L cell membranes available from BioSignal Packard.
Combined with Alpha Screen technology, this provides a rapid , homogeneous, sensitive assay without the variability or time consuming
preparation of whole cells. Assays can be run on demand without waiting
for the preparation of cells; membranes are stored frozen and can be
used whenever needed. Provided here is data using the Dopamine D1
receptor as the membrane preparation and the comparable data expressed
in whole cells.
Figure 4. Principles of cAMP with AlphaScreen
Figure 2. Principles of Alpha Screen [continued]
Figure 1. Principles of A LPHASCREEN
Figure 3. A LPHAQUEST HTS Microplate Analyzer
When biological interactions bring the Donor and Acceptor beads into
close proximity, reactive oxygen, generated by irradiation of the Donor
beads, initiates a luminescence/fluorescence cascade in the Acceptor
beads. This process leads to a highly amplified signal with output in the
520-620 nm range.
The ALPHAQUEST-HTS is a four-detector instrument, optimized for
ALPHASCREEN chemistry, using highly efficient diode excitation at
680 nm with high performance optics detecting emission at 520-620 nm.
With a 40-plate stacker and bar code reader, the ALPHAQUEST-HTS
reads 96-well plates in under 1 minute, 384-well plates in 2.5 minutes
and 1536-well plates in 8.5 minutes.
Laser irradiation of the Donor beads at 680 nm generates a flow of
short-lived singlet oxygen molecules. When the Acceptor beads are not
in proximity, the reactive oxygen decays and there is no signal.
Figure 5. SKF38393 induced cAMP production by
L cells expressing D1 receptors
Membrane Assay protocol
Buffer 1: Oligo element buffer: 25 mM MgCl2, 375 mM NaCl2, 250 uM
The use of SignalScreen membranes with AlphaScreen reagents,
combined with the rapid reading time of the AlphaQuest Microplate
Analyzer make this assay ideal for High Throughput cAMP assays. The
data represented here shows comparable results between membrane
preparations and the equivalent cell based assay and values commonly
reported in the lterature. As the buffers required for the membrane assay
are optimized, to mimic the cell condition, the assay can be done on
demand. Membranes can be stored frozen and used on any day of the
week or month as time permits. They are not subject to the changes or
variation seen in cell culture requiring assays. The current list of
SignalScreen membranes expressing GPCR’s, as well as a complete list
of currently available AlphaScreen reagents can be obtained from
Packard BioSciences.
ATP, 2.5 µM GDP, 2.5 nM GTP. All dissolved in water.
Figure 6. SKF38393 induced cAMP production by
L cell membrane preparation expressing D1 receptors
ALPHASCREEN cAMP has been designed to directly measure levels
of cAMP produced upon modulation of adenylate cyclase activity by
G-protein coupled receptors. ALPHASCREEN cAMP is based on the
competition between endogenous cAMP and exogenously added biotincAMP. The capture of cAMP is achieved by using a specific antibody
conjugated to Acceptor beads. The assay is efficient at measuring both
agonist and antagonist activities on Gi and Gs coupled GPCRs.
ALPHASCREEN cAMP is specific and reliable. This assay is highly
competitive with existing cAMP assays in terms of ease of use, sensitivity, dynamic range and time to completion.
Buffer 2: Lysis buffer (also used for cell based assay): 5 mM Hepes,
pH 7.4, 0.1% BSA, 0.3% Tween 20.
4.3 nM
3.7 nM
Assay Protocol:
10 µl of cAMP acceptor beads (15 µg/ml final concentration in oligo
element buffer)
10 µl membranes (3 µg/well, product # 6100526)
5 µl SKF38393 (forskolin, or unlabeled cAMP for standard curves would
be added at this amount)
Incubate 30 minutes at Room temperature.
Add 25 µl of Streptavidin donor beads (20 µg/ml final in lysis buffer),
premixed with biotin cAMP (10 nM final concentration)
Incubate 1 hour at Room temperature.
Read on AlphaQuest.
Protocol as per Technical Note on Functional cAMP assays from
BioSignal Packard or Packard BioSciences. Application Note also
available at
BioSignal Packard Inc., 1744 William St., Suite 600, Montreal (Quebec), H3J 1R4
Packard Instrument Company, 800 Research Parkway, Meriden, Connecticut 06450