Technical Manual
Transcreener™ PDE Assay
Transcreener™ PDE Assay Kit
Catalog #3006-1K, 3006-10K
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10.0
Introduction
Assay Principle
Instrumentation
Transcreener™ PDE Assay Kit Components
Additional Materials Required
Sample Protocol
AMP/GMP Detection Considerations
Antibody Titration
Standard Curves
User Notification
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1.0 Introduction
Phosphodiesterases control a variety of cellular processes by hydrolyzing the
second messenger signaling molecules cAMP and cGMP. The Transcreener™ PDE Assay
is a far-red, competitive fluorescence polarization immunoassay based on the detection of the hydrolyzed substrates AMP or GMP. Enzyme reaction progress is indicated
by a decrease in the fluorescence polarization signal. The Transcreener™ PDE Assay is
a simple two-step, endpoint assay (Fig. 1) accomodating cAMP/cGMP concentrations
of 0.1 to 10 μM with a single reagent mix (up to 1 mM substrate has been used with Ab
optimization). The assay provides an excellent signal under initial velocity conditions
resulting in overall Z’> 0.6. Although originally designed for PDEs that hydrolyze cAMP
or cGMP, the Transcreener™ PDE Assay can be use for any enzyme class that produces
either AMP or GMP. Reduced development costs and accelerated drug discovery are
achieved by utilizing this one simple streamlined screening method.
Figure 1. Transcreener™ PDE Assay Overview
Enzyme Reaction
AMP/GMP Detection
Add 10 µL enzyme reaction
to assay plate for the desired
time.
Add 10 µL Transcreener PDE
Detection Mix, incubate
1 hour and measure
fluorescence polarization.
2.0 Assay Principle
The Transcreener™ AMP/GMP Detection Mixture comprises an AMP/GMP Alexa633
tracer bound to an AMP/GMP antibody, which is displaced by AMP or GMP, the products generated during PDE reactions. The displaced tracer freely rotates leading to a
decrease in fluorescence polarization, relative to the bound tracer. Therefore, AMP/
GMP production creates a proportional decrease in polarization values. (Fig 2).
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Figure 2. Transcreener™ PDE Assay Principle
3.0 Instrumentation
A microplate reader configured to read fluorescence polarization of Alexa Fluor® 633 is
required. Use standared Cy5 dye instrument configuration. During the development
of this assay kit the following instruments were extensively used and validated: Tecan™
Ultra and Tecan Safire2. Other instruments previously demonstrated effective for
measuring far red fluorescence polarization include: BioTek’s Synergy 2, BMG Labtech’s
PHERAstar and POLARstar, Molecular Devices Analyst (AD, HT, and GT), Perkin Elmer’s
EnVision, ViewLux, and Victor 3V and Tecan’s F500 and GENios Pro. While BellBrook
Labs continues to gather customer feedback regarding instrument set-up and assay
performance it may be necessary to consult the manufacturer’s instructions or technical service representative for instrument set-up to measure fluorescence polarization.
Kit Components
4.0 Transcreener™ PDE Assay Kit Components
Kit Part Number
3006-1K
3006-10K
Number of 20 μL reactions
1,000 Assays
10,000 Assays
AMP/GMP Alexa633 Tracer, 100X
2025 (100 μL)
2030 (1mL)
AMP/GMP Antibody
2026
2031
For antibody concentration, see the certificate of analysis
Stop & Detect Buffer B, 10X
2027 (1 mL)
2032 (10 mL)
500 μM AMP
2028 (500 μL)
2033 (5 mL)
500 μM GMP
2029 (500 μL)
2034 (5 mL)
Store reagents at -20°C. Individual reagents tolerate 10 freeze-thaw cycles.
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5.0 Additional Materials Required
5.1 Enzyme Reaction Mix
The enzyme reaction components supplied by the end-user include enzyme, enzyme
buffer, cAMP and/or cGMP, MgCl2, and test compounds.
5.2 Assay Plate
Recommended plates are Corning® 384 Plates, part number 3676 (black, round bottom, low volume, polystyrene, non-binding surface microplates). It is important to use
completely black plates with low to medium binding.
5.3 Plate Reader
A fluorescence polarization plate reader configured to measure fluorescence polarization of Alexa Fluor® 633 fluorophores. See BellBrook Labs website for recommended
instruments and filters.
5.4 Liquid Handling Devices
The user will need liquid handling devices capable of accurately dispensing volumes of
2.5 to 10 μL into 384-well plates.
6.0 Sample Protocol
The Transcreener™ PDE Assay was designed as a simple two-step endpoint assay that
consists of an enzyme reaction (10 μL) followed by the addition of the AMP/GMP
Detection Mixture (10 μL) for a final volume of 20 μL. AMP/GMP detection sample data
are attached in section 8.0.
6.1 Perform Enzyme Reaction
The antibody concentration on the certificate of analysis is optimized for standard
initial velocity conditions (eg. 10% conversion of 1 μM substrate).
Add enzyme reaction mix to test compounds and mix on plate shaker.
Start the reaction by adding cAMP or cGMP, bringing total volumes to 10 μL. Mix on plate shaker.
Incubate at temperature and time ideal for enzyme target.
6.2 AMP/GMP Detection
Add 10 μL of the AMP/GMP detection mixture and mix on plate shaker.
Incubate at room temperature (20-25 °C) for 1 hour and measure
fluorescence polarization.
7.0 AMP/GMP Detection Considerations
The Transcreener PDE Assay relies on the detection of AMP/GMP in a competitive
fluorescence polarization immunoassay using an AMP/GMP antibody, an AMP/GMP
Alexa633 tracer and a buffer system that stabilizes the fluorescence polarization signal.
This assay was optimized for initial velocity conditions (10-30% conversion) using 0.110 μM substrate.
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7.1 AMP/GMP Antibody
See certificate of analysis for recommended antibody concentrations of purified rabbit
polyclonal anti-AMP/GMP antibody in PBS. A single antibody concentration is suggested for initial cAMP/cGMP concentrations from 0.1 to 10 µM; antibody optimization
is recommended for substrate concentrations outside this range (section 7).
7.2 AMP/GMP Alexa633 Tracer, 100 X
The AMP/GMP Alexa633 Tracer is provided at a concentration of 400 nM in 2 mM
HEPES, pH 7.5 containing 0.01% Brij-35.
7.3 Stop & Detect Buffer B, 10X
Stop & Detect Buffer B (10X) consists of 200 mM HEPES (pH 7.5), 400 mM EDTA, and
0.2% Brij-35. 1X Stop & Detect Buffer B components will stop the enzyme reaction and
aid in the detection and stabilization of the FP signal. EDTA chelates available MgCl2,
preventing further enzyme turnover. The [MgCl2 ] in the enzyme reaction should not
exceed 20 mM to assure sufficient chelation.
7.4 AMP/GMP Detection Mixture
The AMP/GMP Detection Mixture contains AMP/GMP Antibody (see Certificate of
Analysis), 1X AMP/GMP Alexa633 Tracer in 1X Stop & Detect Buffer B.
7.5 AMP/GMP Detection Controls
These controls are used to calibrate the fluorescence polarization plate reader. Both of
these controls are run in the “No PDE” reaction conditions.
7.5.1 No Ab (free tracer) Control: This is the reference sample for the tracer and is set to
20 mP by G-factor calibration.
7.5.2 No Tracer Control: This control is the reference blank for the ‘No Ab’ Control and
the sample blank for all other wells.
7.6 Equilibration
After addition of AMP/GMP Detection Mixture, mix the plate and equilibrate at room
temperature at least 1 hour.
7.7 Measuring Fluorescence Polarization
Configure the FP plate reader to measure the fluorescence polarization of the far-red
Alexa Fluor® 633 fluorophore. A Tecan Safire2 spectrofluorometer was used for validation at BellBrook Labs using Ex635nm LED and Em670nm.
8.0 Antibody Titration
To maximize the assay window and sensitivity, an antibody titration is recommended
in the buffer system ideal for your enzyme target.
8.1 AMP/GMP Antibody Titration
Prepare your enzyme reaction mixture (include cAMP/cGMP, no enzyme) with and
without AMP/GMP antibody (1 mg/mL). Dispense 20 μL of mixture with antibody into
wells in column 1. Dispense 10 μL of the mixture without antibody across a 384-well
plate (columns 2-24). Remove 10 μL from column 1 and serially titrate the contents
across the plate (to column 24).
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8.2 AMP/GMP Detection (No Ab)
Prepare a 1X Detection Mixture with the 100X tracer and the 10X Stop & Detect Buffer
B. Add 10 μL of this mix to the titrated antibody. Mix the plate, equilibrate at room
temperature (at least 1 hour), and measure fluorescence polarization.
8.3 EC85 Determination
The concentration of antibody that is equal to the EC85 value is recommended because
it provides an excellent balance between maximum assay window and sensitivity. Fit
data using a sigmoidal dose (variable slope) response algorithm. The equation for
determining the EC85 value is shown below (using the EC50 and hillslope from the curve
fit).
EC85 = ((85/(100-85))1/hillslope)* EC50.
9.0 AMP/cAMP or GMP/cGMP Standard Curve
When creating a Standard Curve, the total nucleotide concentration should remain
constant. The standard curve below mimics a PDE reaction (as AMP/GMP is produced,
cAMP/cGMP is depleted).
9.1 Prepare Stock Solutions
Using the reaction conditions for your enzyme target, prepare an AMP or GMP stock
solution and cAMP or cGMP stock solution.
9.2 Prepare Standard Curve
Add 10 µL of each standard to the well of a 384 well plate. An example of a twelvepoint AMP/cAMP or GMP/cGMP standard curve mimicking a PDE reaction with 1.0 µM
substrate follows.
12 Point Standard Curve
Percent Substrate Conversion
[AMP] or [GMP] µM
[cAMP] or [cGMP] µM
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0.000
1.000
1.0
0.010
0.990
2.5
0.025
0.975
5.0
0.050
0.950
7.5
0.075
0.925
10.0
0.100
0.900
12.5
0.125
0.875
15.0
0.150
0.850
25.0
0.250
0.750
50.0
0.500
0.500
75.0
0.750
0.250
100.0
1.000
0.000
9.3 Prepare Detection Mixture
Prepare 1X AMP/GMP Detection Mixture (10 μL/well) with an antibody concentration
that is twice the EC85 concentration determined in Section 7.0. NOTE: After adding 10
μL of the 1X AMP/GMP Detection Mixture to the 1X enzyme reaction (10 μL/well) , the
concentration of the antibody will be at the EC85 concentration in 20 μL.
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9.4 Measure FP
Mix components, incubate for 1 hour at room temperature, and measure fluorescence
polarization.
9.5 Plot Standard Curve
Below is sample data for 2 standard curves with initial substrate (cAMP and cGMP)
concentrations of 1.0 µM in the 10 µL enzyme reaction. These standard curves are
designed to mimic the 2-step, end point assay procedure in which 10 µl of 1X Detection Mix is added to a 10 µL enzyme reaction. The nucleotide concentrations indicated
for each point on the standard curve reflect those in the enzyme reaction not those at
time of FP measurement.
Twenty four replicates were performed per conversion level in 25 mM HEPES pH 7.5,
5 mM MgCl2, 1 mM EGTA, 0.5% DMSO, 0.5X Stop & Detect Buffer, 2 nM AMP/GMP
Alexa633 Tracer, and 3.5 µg/mL AMP/GMP Antibody.
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This Transcreener™ PDE Assay is optimized for the initial velocity region. If intended
use is outside the initial velocity region please contact BellBrook Labs for reformatting
recommendations.
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10.0 User Notification
U.S. and foreign patents applied. The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and components of the product in
research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using
this product or its components to a third party or otherwise use this product or its components
or materials made using this product or its components for Commercial Purposes. The buyer may
transfer information or materials made through the use of this product to a scientific collaborator,
provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees
in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1)
use of the product or its components in manufacturing; (2) use of the product or its components
to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether
or not such product or its components are resold for use in research. BellBrook Labs LLC will not
assert a claim against the buyer of infringement of the above patents based upon the manufacture, use, or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed
in research by the buyer in which this product or its components was employed, provided that
neither this product nor any of its components was used in the manufacture of such product. If
the purchaser is not willing to accept the limitations of this limited use statement, BellBrook Labs
LLC is willing to accept return of the product with a full refund. For information on purchasing a
license to this product for purposes other than research, contact Licensing Department, BellBrook
Labs LLC, 525 Science Drive, Suite 110, Madison, Wisconsin 53711. Phone (608)443-2400. Fax
(608)441-2967.
Transcreener™ is a trademark of BellBrook Labs. AlexaFluor® is a registered trademark of Molecular
Probes, Inc (Invitrogen). Corning® is a registered trademark of Corning Incorporated.
Transcreener™ HTS Assay Platform technology is patent pending.
©2006 BellBrook Labs. All rights reserved.
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