cAMP Assay Kit

AlphaScreen® Research Reagents
Caution: For Laboratory Use. A research reagent for research purposes only
cAMP Assay Kit
Product No.: 6760625D/M/R
Lot No.: 1666406
Material Provided
1000 Assay Points
10000 Assay Points
50000 Assay Points
Note: The number of assay points is based on a final bead concentration of 1 unit/well
in a 25 µL/well reaction volume.
Manufacturing Date:
August 27, 2012
Kit Components:
Anti-cAMP Acceptor Beads at 10 U/µL 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
Biotinylated-cAMP at 133 U/µL in TE (10 mM TrisHCl, 1 mM EDTA), 0.05% Proclin-300, pH 8,0
1 x 100 µL
1 x 1 mL
1 x 5 mL
1 x 100 µL
1 x 1 mL
1 x 5 mL
1 x 80 µL
1 x 80 µL
1 x 380 µL
cAMP standard at 50 µM
1 x 1 mL
1 x 1 mL
1 x 1 mL
3% Tween-20
1 x 150 µL
1 x 150 µL
1 x 150 µL
10x Buffer : 50 mM Hepes, 500 mM NaCl, 0.05%
Proclin-300, pH 7.4
1 x 1.5 mL
1 x 1.5 mL
1 x 1.5 mL
Product Information
The cAMP antibody is a goat polyclonal antibody highly specific for cyclic AMP. Its
cross-reactivity with cGMP is < 0.005%.
This product is stable for at least 12 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
20 500 cps
463 cps
14.88 nM
Recommended Assay Conditions
Note: This protocol provides a method to verify kit performance and is not representative of an assay. Sufficient
biotinylated-probe 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). Add 0.05 mL tween20 3% and adjust pH to 7.4.
Acceptor Beads:
Add 5 μL Anti-cAMP Acceptor beads to 245 μL 1x buffer.
Donor beads:
Add 5 μL Streptavidin Donor beads to 730 μL 1x buffer.
Add 5 μL biotinylated-cAMP to 195 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 5 μL of Anti-cAMP Acceptor beads.
Add 5 μL cAMP standard dilutions (from lowest to highest concentration).
Pre-mix the biotinylated-cAMP and Streptavidin Donor beads from previously diluted
solutions by adding 15 µL of biotinylated-cAMP to the Streptavidin Donor beads dilution.
Incubate the premix and the plate in the dark at room temperature for 30 minutes.
Add 15 μL of premix biotinylated-cAMP/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
AlphaScreen signal
log [cAMP] (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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