cAMP AlphaScreen Assay Kit

AlphaScreen® Research Reagents
Caution: For Laboratory Use. A research reagent for research purposes only
cAMP AlphaScreen Assay Kit
Product No.: 6760635D/M/R
Lot No.: 2021213
Material Provided
1000 Assay Points
10000 Assay Points
50000 Assay Points
Note: The number of assay points is based on a final bead concentration in a
25 µL/well reaction volume.
Manufacturing Date:
April 16, 2015
Kit Components:
Anti-cAMP Acceptor Beads at 5 mg/mL in 1X PBS,
0.05% Proclin-300, pH 7.2
Streptavidin Donor Beads at 5 mg/mL in 1X PBS,
0.05% Proclin-300, pH 7.2
1 x 100 µL
1 x 1 mL
1 x 5 mL
1 x 100 µL
1 x 1 mL
1 x 5 mL
Biotinylated-cAMP Tracer Solid
1 x 10 nmol
1 x 10 nmol
1 x 65 nmol
cAMP standard at 50 µM
1 x 1 mL
1 x 1 mL
1 x 1 mL
10x Buffer : AlphaLISA Immunoassay Buffer**
1 x 2 mL
1 x 10 mL
1 x 100 mL
Extra buffer can be ordered separately (cat # AL000C: 10 mL, cat # AL000F: 100 mL).
1X PBS ph 7.4 is not included. It can be ordered from Gibco (cat# 10010-023)
Product Information
The cAMP antibody is a rabbit monoclonal antibody highly specific for cyclic AMP. Its
cross-reactivity with cGMP is < 0.005%.
This product is stable for at least 6 months from the manufacturing date if used and
stored under recommended conditions.
Storage Conditions:
Store undiluted at 4°C protected from light. Freeze-thaw is not recommended and
can cause the beads to form aggregates.
Recommended use:
AlphaScreen® Donor beads are light-sensitive. All Alpha assays using the Donor
beads should be performed under subdued laboratory lighting (< 100 lux). Green
filters (LEE 090 filters (preferred) or Roscolux filters #389 from Rosco) can be
applied to light fixtures.
Page 1 of 3
For additional information on running AlphaScreen assays or on potential interfering compounds, please visit our
Quality Control
AlphaScreen maximum signal, minimum signal and IC50 are determined by a cAMP competition assay performed
on an EnVision HTS Alpha detection instrument. We certify that these results meet our requirements.
Maximum signal
Minimum signal
LDL (nM)
IC50 (nM)
Recommended Assay Conditions
Note: This protocol provides a method to verify kit performance and is not representative of an assay. Sufficient
biotinylated cAMP-Tracer and 10x buffer is provided to perform 3 competition curves in triplicate as described.
1x Buffer:
Add 500 μL 10x buffer to 4.45 mL Milli-Q H2O (or equivalent).
Acceptor Beads:
Add 5 μL Anti-cAMP Acceptor beads to 495 μL 1x buffer.
biotinylated cAMP-Tracer:
Dissolve biotinylated cAMP-Tracer (10 nmol) in 1 mL 1X PBS (final 10 μM)
Further dilute 10X in 1 X PBS, pH 7.4 to obtain a 1 M solution.
*Solids may not be visible deposited along the walls of the tube*
Donor beads:
Add 6.25 μL Streptavidin Donor beads to 579.75 μL 1x buffer.
cAMP standard:
From the 50 µM cAMP standard, prepare a ½ log dilution series (5 µM to 50 pM) in
1x buffer. Include a buffer only control.
Titration Protocol:
To a white opaque 384-well Optiplate:
Add 10 μL of Anti-cAMP Acceptor beads.
Add 5 μL cAMP standard dilutions (from lowest to highest concentration).
Pre-mix the biotinylated cAMP-Tracer and Streptavidin Donor beads from previously diluted
solutions by adding 39 µL of biotinylated cAMP-Tracer to the Streptavidin Donor beads
Incubate the premix and the plate in the dark at room temperature for 30 minutes.
Add 10 μL of premix biotinylated cAMP-Tracer /Streptavidin Donor beads.
Incubate in the dark at room temperature for 60 minutes and analyze on your AlphaScreen detection
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Product Typical Data
A lp h a S c r e e n S ig n a l (c o u n t s )
c A M P A lp h a S c r e e n
-1 4
-1 2
-1 0
[c A M P ] ( M )
Figure 1: Competition assay
384-well cAMP competition curve (25 μL final volume;
Reader: Envision HTS Alpha).
Note: AlphaScreen signal will vary depending on
instrument detection protocol, incubation temperature
and incubation time.
Suggested Materials and Instrumentation
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This product is not for resale or distribution except by authorized distributors.
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