Transcreener PDE Assay

Transcription

1 Technical Manual Transcreener PDE Assay

2 Transcreener PDE Assay Kit Catalog #3006-1K, K 1.0 Introduction p Assay Principle p Instrumentation p Transcreener PDE Assay Kit Components p Additional Materials Required p Sample Protocol p AMP/GMP Detection Considerations p Antibody Titration p Standard Curves p User Notification p 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 Add 10 µl enzyme reaction to assay plate for the desired time. AMP/GMP Detection 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). p

3 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 Safire 2. 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. 4.0 Transcreener PDE Assay Kit Components Kit Components Kit Part Number K K Number of 20 μl reactions 1,000 Assays 10,000 Assays AMP/GMP Alexa633 Tracer, 100X 2025 (100 μl) 2030 (1mL) AMP/GMP Antibody 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. p.3

4 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, MgCl 2, 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 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 μm substrate. p

5 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 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 MgCl 2, preventing further enzyme turnover. The [MgCl 2 ] 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 No Ab (free tracer) Control: This is the reference sample for the tracer and is set to 20 mp by G-factor calibration 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 Safire 2 spectrofluorometer was used for validation at BellBrook Labs using Ex 635nm LED and Em 670nm. 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). p.5

6 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 EC 85 Determination The concentration of antibody that is equal to the EC 85 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 EC 85 value is shown below (using the EC 50 and hillslope from the curve fit). EC 85 = ((85/(100-85)) 1/hillslope )* EC 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 Prepare Detection Mixture Prepare 1X AMP/GMP Detection Mixture (10 μl/well) with an antibody concentration that is twice the EC 85 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 EC 85 concentration in 20 μl. p

7 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 MgCl 2, 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. expanded 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. p.7

8 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 Phone (608) Fax (608) 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 BellBrook Labs. All rights reserved. p.8 v091907