Technical Manual Transcreener KINASE TR-FRET Assay
Transcreener KINASE TR-FRET Assay Kit Instructions for Part Numbers 3005-1K and 3005-10K 1.0 Introduction p.2 2.0 Assay Principle p.3 3.0 Assay Components p.3 4.0 Additional Materials Required p.3 5.0 Enzyme Assay Considerations p.4 6.0 ADP Detection Considerations p.5 7.0 ADP FAM Tracer Titration p.6 8.0 ADP/ATP Standard Curve p.6 9.0 Signal Stability p.8 10.0 User Notification p.8 1.0 Introduction The Transcreener HTS assay platform is a universal, homogenous, high throughput screening (HTS) technology developed to monitor the activity of enzymes that catalyze group transfer reactions. Group transfer enzyme families include kinases, UDP-glycosyltransferases (UGTs), sulfotransferases, methyltransferases, acetyltransferases, and ADP-ribosyltransferases. For most group transfer reactions, an activated nucleotide serves as the donor, and the acceptor can be a protein, lipid, carbohydrate, or nucleic acid. The products of the group transfer reactions are the conjugated acceptor and an invariant donor by-product. The Transcreener HTS technology overcomes the need for time-consuming, one-off assay development for individual members within a group transfer enzyme family by utilizing a single set of assay reagents that detect this invariant product. The generic nature of the Transcreener platform will eliminate delays involved in assay development for new HTS targets, and will greatly simplify compound and inhibitor profiling across multiple family members. The Transcreener KINASE TR-FRET Assay is a competitive, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay for detection of ADP. The Transcreener KINASE TR-FRET Assay can be used for any enzyme class that produces ADP, including kinases, ATPases, DNA helicases, and acetyl CoA carboxylases. Kinases catalyze the transfer of a phosphate group from ATP to a protein, peptide, lipid, or small molecule substrate generating ADP, the invariant product. For ATPases, a phosphate group from ATP is transferred to a water molecule. The Transcreener KINASE TR-FRET Assay was designed as a simple two-step, endpoint assay (a 10 µl enzyme reaction followed by addition of 10 µl ADP Detection Mixture (Figure 1). The assay provides an excellent signal at low ATP conversion resulting in overall Z > 0.6. The Transcreener KINASE TR-FRET Assay was developed for optimal performance at 10% ATP conversion and is flexible with regard to ATP concentration (from 1 µm to 100 µm ATP). Figure 1. Transcreener KINASE TR-FRET Assay overview Enzyme Reaction Incubate 10 µl enzyme reaction in assay plate for desired time. ADP Detection Add 10 µl Transcreener ADP Detection Mix, incubate 1 hour and read TR-FRET. p.2 866.313.7881
2.0 Assay Principle The Transcreener KINASE TR-FRET Assay measures ADP in a biomolecular competitive immunoassay using a proprietary monoclonal ADP Antibody conjugated to a terbium chelate (ADP Antibody-Tb) and an ADP FAM Tracer. TR-FRET is observed when the lanthanide donor (ADP Antibody-Tb) is bound to the small molecule fluorescein acceptor (ADP FAM Tracer). ADP generated during the enzyme reaction displaces the ADP-FAM Tracer from the ADP Antibody-Tb disrupting TR-FRET. Lanthanide molecules (terbium, europium) have long fluorescence lifetimes. Measuring the FRET signal in a time-gated mode reduces autofluorescent interference of some test compounds, which typically have shorter fluorescent lifetimes. The TR-FRET ratiometric read-out (FAM 520nm /Tb 495nm ) limits fluctuations in signal variability caused by interwell effects in sample turbidity and reagent volumes (Figure 2). Figure 2. Transcreener KINASE TR-FRET Assay Principle + + Phosphorylated Substrate + 3.0 Assay Components Part Numbers for the Transcreener KINASE TR-FRET Assay. Kit Component 3005-1K part number (volume) 3005-10K part number (volume) ADP FAM Tracer, 5 µm 2019 (160 µl) 2022 (1.6 ml) ADP Antibody-Tb, 100X* 2020 (100 µl) 2023 (1 ml) Stop & Detect Buffer A, 10X 2021 (1 ml) 2024 (10 ml) 500 µm ADP 2012 (500 µl) 2016 (5 ml) Store individual reagents at -80 C. *The ADP Antibody-Tb, 100X is sensitive to multiple freeze/thaw cycles. To maintain a robust assay, aliquot the ADP Antibody-Tb, 100X after the initial thaw. The ADP FAM Tracer (5 µm), Stop & Detect Buffer A, 10X, and 500 µm ADP can withstand >5 freeze/thaw cycles. 4.0 Additional Materials Required 4.1 Enzyme Reaction Components The enzyme reaction components supplied by the end-user include enzyme, enzyme buffer, acceptor substrate, ATP, MgCl 2, EGTA, Brij-35, and test compounds. www.bellbrooklabs.com p.3
4.2 Assay Plate Recommended plates are Corning white (catalog # 3673) or black (catalog # 3676) 384-well, round bottom, low volume, polystyrene, non-binding surface plates. 4.3 Plate Reader A plate reader configured to measure time-resolved fluorescence resonance transfer (TR-FRET) is required. The Transcreener KINASE TR-FRET Assay has been successfully used on both filter-based (Tecan Ultra) and monochromator-based (Tecan Safire 2 ) instruments. Filter sets were Ex 340nm (bw = 35 nm), Em 495nm (bw = 10 nm), Em 520nm (bw = 25 nm). Tecan Ultra and Tecan Safire 2TM settings included a 100 µsec delay, 100-200 µsec integration time, 20 flashes at 30 C. Suitable excitation and emission filters can be purchased from Chroma Technology Corp. (Part Numbers PV002 and PV003). A description of instruments compatible TR-FRET (Tecan Ultra and GENios Pro, Molecular Devices (LJL) Analyst HT, Perkin Elmer EnVision, and BMG LABTECH PHERAstar) can be found at http://www.invitrogen.com. 4.4 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. 5.0 Enzyme Assay Considerations The Transcreener KINASE TR-FRET Assay is a universal biochemical assay designed for enzymes that produce ADP. The enzyme reaction can be performed at any temperature and for any length of reaction. The ADP detection reagents are not added until after the user-defined enzyme reaction is complete. 5.1 Enzyme Assay Conditions The Transcreener KINASE TR-FRET Assay components have been optimized for endpoint measurement of enzyme reactions containing 50 mm HEPES (ph 7.5), 4 mm MgCl 2, 2 mm EGTA, 1% DMSO (test compound solvent), 0.01% Brij-35 and varying ATP concentrations (1 µm to 100 µm ATP). However, buffer components ideal for any enzyme target can be used. To produce a sensitive and robust assay signal, performing a tracer titration in the buffer system ideal for your enzyme target is recommended (See Section 7.0). Note: In the two-step protocol, the concentration of the enzyme reagents (including ATP) after addition of the ADP detection reagents is 0.5X. 5.2 Enzyme Assay Controls 5.2.1 0% ATP Conversion (No Enzyme) Control: This control consists of the ADP Antibody-Tb and ADP FAM Tracer complex in the presence of enzyme reaction components and 100% ATP (0% ADP) without enzyme. This control defines the upper limit of the assay window. 5.2.2 100% ATP Conversion Control: This control consists of the ADP Antibody-Tb and ADP FAM Tracer complex in the presence of enzyme reaction components and 100% ADP (0% ATP) without enzyme. This control defines the lower limit of the assay window. 5.2.3 No Acceptor Substrate Control: This control consists of the ADP Antibody-Tb and ADP FAM Tracer complex in the presence of enzyme reaction components and 100% ATP (0% ADP) without substrate. Because some enzymes show activity in the absence p.4 866.313.7881
of acceptor substrate, this control should not be used to define the upper limit of the assay window. However, this control can be used to monitor substrate-independent ADP production. 5.2.4 ADP/ATP Standard Curve: During the enzyme reaction, as ADP is produced, ATP is depleted. By graphing the TR-FRET ratio (520 nm/495 nm) vs µm ADP, the TR-FRET ratio can be correlated to the concentration of ADP generated (and then to % ATP conversion). See section 8.0 for a detailed description for preparing an ADP/ATP standard curve. 5.3 Endpoint Assay The Transcreener KINASE TR-FRET Assay is designed for an endpoint readout. The Stop & Detect Buffer A, 10X contains EDTA to stabilize the signal. EDTA stops the enzyme reaction by chelating available MgCl 2, which is required for enzyme turnover. 5.4 Realtime Assay The end-user may perform kinetic experiments by substituting the Stop & Detect Buffer A, 10X (provided) with a preferred detection buffer (prepared by the user without EDTA). See Section 6.1 for the Stop & Detect Buffer A, 10X recipe. Note: depending on the other reaction components, optimal [ADP FAM Tracer] may change when EDTA is omitted (See Section 7.0 for Tracer titration). 6.0 ADP Detection Considerations The Transcreener KINASE TR-FRET Assay can accommodate a wide range of ATP concentrations. See the certificate of analysis for recommended ADP FAM Tracer concentrations at 1 µm, 10 µm, 50 µm, and 100 µm ATP using a common buffer (Section 5.1). 6.1 Stop & Detect Buffer A, 10X The TR-FRET Stop & Detect Buffer A (10X) consists of 500 mm HEPES (ph 7.5), 2 M NaCl, 50 mm EDTA, and 0.2% Brij-35. The 1X Stop & Detect Buffer A components will stop the enzyme reaction. To ensure the enzyme reaction is stopped completely, confirm that the EDTA concentration provided is at equimolar relative to the Mg +2 ions present. 6.2 ADP Antibody-Tb, 100X BellBrook Labs proprietary monoclonal ADP Antibody has been conjugated to the Lanthascreen Terbium Chelate and is supplied at a 100X concentration (400 nm) in 25 mm HEPES (ph 7.5), 137 mm NaCl, 5 mm KCl, and 1 mm Na 2 HPO 4. 6.3 ADP FAM Tracer, 5 µm The ADP FAM Tracer consists of a fluorescein conjugated to ADP and is supplied in 2 mm HEPES (ph 7.5) and 0.01% Brij-35. 6.4 1X Stop and Detect Reagents 1X Stop and Detect Mixture (10 µl) is added to 1X enzyme reaction (10 µl) resulting in 0.5X of each component in the final 20 µl volume. The 1X Stop and Detect Mixture consists of 1X ADP Antibody-Tb and ADP FAM Tracer in 1X Stop and Detect Buffer A. The optimal concentration of ADP FAM Tracer is dependent upon the buffer components (including [ATP]) in the enzyme reaction. See Section 7 to determine the optimal [ADP FAM Tracer] for your assay. www.bellbrooklabs.com p.5
6.5 ADP Detection Controls These controls identify background signal and are used to establish the TR-FRET signal in the absence of one of the fluorescent probes. 6.5.1 No Ab (free tracer) Control: This sample contains the ADP FAM Tracer without the ADP Antibody-Tb. 6.5.2 No Tracer Control: This sample contains the ADP Antibody-Tb without the ADP FAM Tracer. 6.6 Measuring TR-FRET Both the fluorescein signal (520nm) and the terbium signal (495) are read in a timegated mode. The TR-FRET ratiometric readout (520nm/495nm) provides signal stability. 7.0 ADP FAM Tracer Titration To produce a sensitive and robust assay signal, performing a tracer titration in the buffer system ideal for your enzyme target is recommended. A brief description follows. 7.1 Perform a 2-fold serial dilution of ADP FAM Tracer in your enzyme reaction mixture. Prepare your enzyme reaction mixture (include substrate and ATP) with and without ADP FAM Tracer (250 nm). Dispense 20 µl of mixture with ADP FAM Tracer into wells in row 1. Dispense 10 µl of the mixture without ADP FAM Tracer across a 384-well plate (rows 2-24). Remove 10 µl from row 1 and perform a two-fold serial titration across the plate (to row 24). 7.2 Add ADP Antibody-Tb and Measure TR-FRET. Prepare 1X ADP Antibody-Tb in 1X Stop and Detect Buffer A and add 10 µl to the titrated tracer. Mix the plate, equilibrate at room temperature (1 hour), and measure TR-FRET. 7.3 Plot [ADP FAM Tracer] vs TR-FRET ratio (520nm/495 nm) on a log scale. To produce a signal that is both sensitive and robust, the EC 50 concentration of ADP FAM Tracer is recommended. Graph data using sigmoidal dose response curve fit. 8.0 ADP/ATP Standard Curve This standard curve mimics a enzyme reaction (as ADP is produced, ATP is depleted). When creating an ADP/ATP Standard Curve, the adenosine concentration should remain constant. 8.1 Prepare stock solutions Prepare an ADP stock solution and ATP stock solution in your enzyme reaction mixture (including substrate). p.6 866.313.7881
8.2 Prepare a twelve-point standard curve Prepare a twelve-point standard curve for a 10 µm ATP enzyme reaction as seen below, and add mixes to the plate. % ATP Conversion ADP (µm)/atp (µm) Concentration Volume of 10 µm ADP/10 µm ATP (final volume = 100 µl) 0 0 µm/ 10.0 µm 0 µl/ 100 µl 0.5 0.05 µm/ 9.95 µm 0.5 µl/ 99.5 µl 1 0.1 µm/ 9.9 µm 1 µl/ 99 µl 2 0.2 µm/ 9.8 µm 2 µl/ 98 µl 4 0.4 µm/ 9.6 µm 4 µl/ 96 µl 6 0.6 µm/ 9.4 µm 6 µl/ 94 µl 8 0.8 µm/ 9.2 µm 8 µl/ 92 µl 10 1.0 µm/ 9.0 µm 10 µl/ 90 µl 20 2.0 µm/ 8.0 µm 20 µl/ 80 µl 30 3.0 µm/ 7.0 µm 30 µl/ 70 µl 50 5.0 µm/ 5.0 µm 50 µl/ 50 µl 100 10.0 µm/ 0 µm 100 µl/ 0 µl 8.3 Prepare 1X ADP Detection Mixture Prepare 1X ADP Detection Mixture (10 µl/well) with an ADP FAM Tracer concentration that is twice the EC 50 concentration determined as in Section 7.0. NOTE: After adding 10 µl of the 1X ADP Detection Mixture to the 1X enzyme reaction (10 µl), the concentration of ADP FAM Tracer will be at the EC 50 concentration (in 20 µl). Mix components on a plate shaker, incubate for 1 hour at room temperature, and measure TR-FRET as in Section 4.3. Figure 3. 10 µm ADP/ATP Standard Curve Data The 10 µm ADP/ATP Standard Curve mimics a typical enzyme reaction (as ADP is produced, ATP is depleted) by keeping the adenosine concentration constant. Graphing on the log scale eliminates the point that corresponds to zero. To include all twelve points in the curve, the 0 µm ADP/10 µm ATP was graphed at the 0.01 µm ADP/9.99 µm ATP position. The Transcreener KINASE TR-FRET Assay provides excellent signal and optimal assay performance at 10% ATP conversion. The Transcreener KINASE TR-FRET Assay can accomodate any [ATP] between 1 µm to 100 µm. www.bellbrooklabs.com p.7
9.0 Signal Stability 9.1 Plate Stability The TR-FRET signal is stable for at least 5 hours at room temperature (20-25 C) after adding the 1X ADP Detection reagents to the plate. 9.2 Deck Stability Deck Stability is a measure of the time the pre-mixed ADP Detection Reagents are stable on the HTS platform. The 1X ADP Detection Mixture is stable for 8 hours at room temperature (20-25 C) before adding to the plate. A loss in signal intensity is observed with incubation overnight, however the effect on the ratiometric signal is minimal. 10.0 User Notification U.S. and foreign patents applied. The purchase of this product conveys to the buyer the non-transferable 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 (866)313-7881. Fax (608)441-2967. Transcreener is a trademark of BellBrook Labs. Lanthascreen is a trademark of Invitrogen. Safire 2TM is a trademark of Tecan. Corning is a registered trademark of Corning Incorporated. Transcreener HTS Assay Platform is patent pending. 2006 BellBrook Labs. All rights reserved. p.8 v082107