FAR RED FP. UDP FP Assay. Technical Manual. v042809

Transcription

1 FAR RED FP UDP FP Assay Technical Manual v042809

2 Transcreener UDP FP Assay Kit Instructions for Part Number K and K 1.0 Introduction p Assay Components p Protocol p.4 Set up UDP Detection p.4 Detect UDP in an Enzyme Reaction p Reagent and Signal Stability p Introduction The Transcreener HTS Assay platform overcomes the need for time-consuming, oneoff assay development for individual members within a group transfer enzyme family by utilizing a single set of assay reagents that detect an invariant product. The generic nature of the Transcreener HTS Assay platform eliminates delays involved in assay development for new HTS targets, and greatly simplifies compound and inhibitor profiling across multiple target families. The Transcreener UDP FP Assay is a competitive fluorescence polarization (FP) assay based on the detection of UDP and therefore is compatible with any enzyme class that produces UDP, including glucosyltransferase, galactosyltransferase, glucuronyltransferase, N-acetylglucosamyltransferase, N-acetylgalactosyltransferase, xylosyltransferases, and glycogen, cellulose, lactose and hyaluronan synthases. The Transcreener UDP FP Assay is a simple one step homogenous detection assay, and is extremely flexible with regard to substrate concentration (1 to 250 µm). The assay provides an excellent signal under initial velocity conditions resulting in an overall Z > 0.6. Figure 1. Transcreener UDP Far Red FP Assay Overview The Transcreener UDP FP Assay was developed to follow the progress of any enzyme that produces UDP. The Transcreener UDP Detection Mixture comprises an UDP DyLight 632 Tracer bound to an UDP Antibody. The tracer is displaced by UDP, the invariant product generated during the enzyme reaction (Figure 1). The displaced tracer freely rotates leading to a decrease in fluorescence polarization. Therefore, UDP production leads to a decrease in polarization. The assay uses a far red tracer to minimize interference from fluorescent compounds and light scattering. p

3 2.0 Transcreener UDP FP Assay Components Store reagents at -20 C with the exception of UDP DyLight 632 Tracer, which should be stored at -80 C. Individual reagents tolerate 10 freeze-thaw cycles. Sufficient reagents are provided to complete the protocol when using substrates within a range of 1 to 100 µm. Please contact BellBrook Labs for custom packaging for enzyme reactions with >100 µm substrate. UDP Antibody A concentrated polyclonal UDP Antibody is provided in PBS. The concentration of UDP Antibody needed for an enzyme target is dependent upon the substrate concentration and buffer conditions in the enzyme reaction. To produce the most sensitive and robust assay signal, it is necessary to perform an UDP Antibody titration in the buffer system ideal for your enzyme target. UDP DyLight 632 Tracer, 400 nm The UDP DyLight 632 Tracer is provided at a concentration of 400 nm in 2 mm HEPES, ph 7.5 containing 0.01% Brij-35. This kit was optimized with 2nM UDP DyLight 632 Tracer in the final 20 μl reaction. 500 mm Na 3 This reagent is included to stabilize UDP in impure enzyme sources, such as Human Liver Microsomes (HLMs). 500 mm Na 3 is added to the UDP Detection Mixture for detection in HLM reactions. 5 mm UDP UDP is not common to all laboratories and therefore is a supplied reagent. UDP is used to create the UDP-sugar/UDP standard curve. 500 mm EGTA Included to stabilize UDP in impure enzyme sources, such as HLMs. 500 mm KPO 4, ph 7.5 Included as the stock buffer for the UDP Detection Mixture. Materials Required but Not Provided Ultrapure Water Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, therefore reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. Enzyme Buffer Components The enzyme reaction components supplied by the end-user include enzyme, reaction buffer, UDP-glycosyl substrate, acceptor substrate and test compounds. The UDPglycosyl substrate should be the highest purity available. Contaminating UDP will decrease the assay sensitivity. Avoid multiple freeze thaws. p.3

4 Plate Reader A microplate reader configured to read fluorescence polarization of DyLight 632 fluorophor is required. Use standard Cy5 dye instrument configuration. During the development of this assay 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. Contact BellBrook Labs for additional information regarding instrument set-up and fluorescence polarization measurements. Assay Plate It is important to use assay plates that are entirely black with a non-binding surface. We recommend Corning 384 plates (catalog #3676). Liquid Handling Devices Use liquid handling devices that can accurately dispense a minimum of 2.5 µl into 384-well plates. 3.0 Protocol The Transcreener UDP FP Assay is designed around the customer s enzyme buffer conditions. The UDP Detection system must be established before running the enzyme assay. These steps include instrument set-up and antibody optimization. Completing these initial steps will provide optimal UDP detection results and need not be repeated unless assay reaction conditions change. Instrument Set Up Antibody Titration Enzyme Reaction + UDP-Sugar /UDP Standard Curve UDP Detection Set up UDP Detection Instrument Settings Becoming familiar with ideal instrument settings for fluorescence polarization is essential to the success of the Transcreener UDP FP Assay. Verify instrument measures fluorescence polarization. Ensure the instrument is capable of measuring fluorescence polarization (not simply fluorescence intensity) of DyLight 632. Please call BellBrook Labs if you have questions about settings and filter sets for a specific instrument. Define the maximum mp window for your instrument. Measuring high (tracer + antibody) and low (free tracer) polarization will define the maximum assay window of your specific instrument. High polarization mixture Prepare 20 μg/ml UDP Antibody in 2 nm UDP DyLight 632 Tracer/10 mm KPO 4, ph 7.5. p

5 Low polarization mixture Prepare 2 nm UDP DyLight 632 Tracer/10 mm KPO 4, ph 7.5 without UDP Antibody. Measure the fluorescence polarization. The difference between the low and high polarization values should be >175 mp. If the assay window is <175 mp, please call BellBrook Labs. Optimize UDP Antibody Concentration The Transcreener UDP FP Assay requires detection of UDP in the presence of excess substrate (assuming initial velocity enzyme reaction conditions) using an antibody with a finite selectivity for the diphosphate vs. the UDP-glycosyl substrate. The concentration of UDP Antibody determines the total assay window and the UDP detection range, and the amount needed is dependent upon the substrate concentration in the enzyme reaction. To produce the most sensitive and robust assay signal, it is necessary to perform an UDP Antibody titration in the buffer system ideal for your enzyme target. To determine the optimal antibody concentration, titrate the UDP Antibody using the reaction conditions for your enzyme or drug target. The nucleotide substrate concentration in the enzyme reaction generally determines the appropriate concentration of UDP Antibody. We recommend using the EC 85 concentration of antibody. Titrate UDP Antibody in 1X Stop & Detect Buffer Prepare your enzyme reaction mixture (include UDP-sugar, 2 nm UDP DyLight 632 Tracer, exclude acceptor substrate) with and without UDP antibody. Dispense 30 μl of mixture with an appropriate starting concentration of antibody into wells in column 1. A common starting antibody concentration is 2 mg/ml. Dispense 15 μl of the mixture without antibody across a 384-well plate (columns 2-24). Remove 15 μl from column 1 and serially titrate the contents across the plate (to column 24). Equilibrate at room temperature for at least 90 minutes, and measure fluorescence polarization. UDP Detection Controls These controls are used to calibrate the fluorescence polarization plate reader controls and are added to wells that do not contain enzyme. Without Antibody (free tracer) Control This sample contains the UDP DyLight 632 Tracer without the UDP Antibody and is set to 20 mp. Without Tracer Control This sample contains the UDP Antibody without the UDP DyLight 632 Tracer and is used as a sample blank for all wells. It is used as a buffer blank, but contains the same UDP Antibody concentration used in all wells. p.5

6 Plot mp vs. log of UDP Antibody concentration and calculate the EC 85 The antibody concentration at the EC 85 is often used as a good compromise between sensitivity and maximal polarization value. The EC 85 is determined by inputting the EC50 and hillslope values from a sigmoidal dose response curve fit into the equation below. EC 85 = ((85/(100-85))1/hillslope)* EC 50 Detect UDP in an Enzyme Reaction The Transcreener UDP FP Assay is a universal biochemical assay designed for enzymes that produce UDP. In the one-step detection protocol, 5 µl UDP Detection Mixture is added to the 15 µl enzyme reaction then mixed and incubated for 1 hour. Figure 2. Protocol Enzyme Reaction 15 μl UDP Detection UDP 5 µl Final reaction volume 20 μl Enzyme Reaction (15 µl) Add the enzyme reaction mixture to test compounds and mix on plate shaker. Start the reaction by adding substrate and mix. Incubate at temperature and time ideal for enzyme target before addition of the UDP Detection Mixture. For a maximum assay window include tracer in the enzyme reaction if possible. The order of addition effects the assay window. UDP Detection Mixture (5 µl) The UDP Detection Mixture is prepared by adding UDP Antibody (4*EC 85 ), 2 nm UDP DyLight 632 Tracer (final concentration; do not include if it is in the enzyme reaction), and 10 mm KPO 4, ph 7.4. For impure enzyme preparations, such as HLMs, include 100 mm Na 3 (25 mm final concentration in the final 20 μl detection reaction) in the UDP Detection Mixture to stabilize the signal molecule, UDP. Add the UDP Detection Mixture to the enzyme reaction and mix on plate shaker. Incubate at room temperature (20-25 C) for 90 minutes, and measure fluorescence polarization. Enzyme Reaction Conditions Choose enzyme buffer conditions and UDP Detection Mixture that are ideal for your enzyme target. Perform your enzymatic reaction at its requisite temperature and optimal duration. An enzyme titration can be performed to identify the optimal enzyme concentration for the Transcreener UDP FP Assay. Quality control of the Transcreener UDP FP Assay components is performed in 50 mm HEPES (ph 7.5), 4 mm MgCl 2, 2 mm EGTA, 1% DMSO (test compound solvent), 0.01% Brij-35 and varying substrate concentrations (10 to 100 µm). p

7 Enzyme Assay Controls The enzyme reaction controls define the limits of the enzyme assay. 0% Substrate Conversion Control This control consists of the UDP Detection Mixture, the enzyme reaction components (without enzyme), and 100% substrate (0% UDP). This control defines the upper limit of the assay window. 100% Substrate Conversion Control This control consists of the UDP Detection Mixture, the enzyme reaction components (without enzyme) and 100% UDP (0% substrate). This control defines the lower limit of the assay window. Without Acceptor Substrate Control This control consists of the UDP Detection Mixture, the enzyme reaction components (without substrate) and 100% substrate (0% UDP). Because some enzymes show activity in the absence 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 UDP production. Without Nucleotide Control To verify that the enzyme does not interfere with the detection module, perform an enzyme titration in the absence of donor and acceptor substrate. UDP-Sugar/UDP Standard Curve The standard curve mimics an enzyme reaction (as UDP-sugar concentration decreases, UDP concentration increases); the uridine concentration remains constant. The UDP-sugar/UDP standard curve allows calculation of the concentration of UDP produced in the enzyme reaction and therefore the % substrate consumed (%UDP-sugar conversion). Prepare a twelve-point curve using concentrations of UDP and UDP-sugar corresponding to 0%, 2%, 4%, 6%, 8%, 10%, 15%, 20%, 30%, 40%, 60%, and 100% substrate conversion. p.7

8 Figure μm UDP/UDPGA Standard Curve of mp data Figure 3 plots the mp data from 24 replicates per percent conversion. To include zero in log scale format, 0.04 was used as the 0% conversion point. The assay was performed in 20 mm KPO 4 ph 7.5, 5 mm MgCl 2, 2 nm UDP DyLight 632 Tracer, and 62.5 μg/ml UDP Antibody. Figure μm UDP/UDPGA Standard Curve mp Data Figure 4 plots mp data obtained from Figure 3. mp = mp initial [UDPGA] - mp sample and Z =1- [(3*SD initial [UDPGA] + 3*SD sample )/ (mp initial [UDPGA] - mp sample ) ] p

9 Endpoint Assay The Transcreener UDP FP Assay is designed for endpoint readout. Na 3 has been developed as a stop reagent for the hepatic UGTs and has not been tested with UGTs outside this class (Na 3 is a nucleotide analog). If a stop reagent other than Na 3 is required please include it during antibody optimization or contact BellBrook Labs for chemical compatibility information. Realtime Assay You may perform real-time experiments by substituting the UDP Detection Mixture with a detection buffer that does not contain NA 3. Note that the optimal UDP Antibody concentration may change when NA 3 is omitted. 4.0 Reagent and Signal Stability The Transcreener technology provides the end-user with a robust and stable assay method to detect UDP. Signal Stability The stability of the mp assay window at 10% substrate conversion was determined after the addition of the UDP Detection Mixture to the standard samples. The mp assay window at 10% substrate conversion (10 µm) remained constant (< 10% change) for at least 24 hours at room temperature (20-25 C). If plates are to be read the following day, they should first be sealed to prevent evaporation. UDP Detection Mixture Stability The UDP Detection Mixture is stable for at least 24 hours at room temperature (20-25 C) before adding to the enzyme reaction (i.e. stored on the liquid handling deck). Solvent Compatibility The mp assay window at 10% substrate conversion (10 µm substrate) remains constant (< 10% change) when up to 10% DMSO, ethanol, or acetonitrile are used in the enzyme reaction. Contact BellBrook Labs for further reagent compatibility information. p.9

10 Patent 7,332,278, 7,355,010 and 7,378,505 issued. U.S. Patent Application Nos. 11/353,500 and 11/958,965 and International Patent Application Nos. PCT/US04/ 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, 5500 Nobel Drive, Suite 250, Madison, Wisconsin Phone (608) Fax (608) Transcreener HTS Assay Platform is a patented technology of BellBrook Labs. Transcreener is a registered trademark of BellBrook Labs. DyLight 632 is a registered trademark of Pierce Biotechnology (Thermo Fisher Scientific) BellBrook Labs. All rights reserved. p