htrf-Kinase TK 20000

www.htrf.com HTRF® package insert For in vitro research use only R&D, Administration and Europe Office Cisbio Bioassays Phone: +33 (0)4 66 79 67 05 Fax: +33 (0)4 66 79 19 20 E-mail: bioassays@cisbio.com Japan Office Sceti Medical Labo K.K. Phone: +81 (0)3 5510 2932 Fax: +81 (0)3 5510 0130 E-mail: reagent@scetimedilabo.co.jp USA Office Cisbio US, Inc. Phone : 888 963 4567 Fax : 781 687 1500 E-mail : htrfinfo@cisbio.us HTRF® KinEASE™-TK 20,000 tests Packaging details: 384-well low volume plate (20 µL) 62TK0PEC HTRF® KinEASE™-TK 20,000 tests Document reference : 62TK0PEC rev04 (September 2009) Storage temperature: 2-8°C 1. Assay description and intended use HTRF® KinEASE™ -TK is a generic method for measuring tyrosine kinase activities using one substrate and a universal detection system. To date, more than 59 kinases have already been tested with this kit (see Appendix). The HTRF® KinEASE™-TK assay format involves the two steps described below: 1. Enzymatic step: During this step, the kinase will phosphorylate the substrate. The TK Substrate-biotin is incubated with the kinase. ATP is added to start the enzymatic reaction. TK Antibody labeled with Eu3+-Cryptate TK Substrate-biotin Kinase ATP TR-FRET EDTA Detection reagents Streptavidin-XL665 2. Detection step: The detection reagents will catch the phosphorylated substrate. The resulting TR-FRET signal is proportional to the phosphorylation level. The TK-Antibody labeled with Eu3+-Cryptate and streptavidin-XL665 are then added with EDTA (used to stop the kinase activity). 2 2. Kit description - Stock solution preparation The kit is designed to allow 20,000 tests to be run at 20 µL final volume, providing that the substrate final concentration is ≤ 1 µM and streptavidin/substrate ratio ≤ 1/4. Components Quantity Stock solutions to prepare TK Substrate -biotin 1 vial of 500 µgLyophilized  Reconstitute with distilled water (refer to product label) to obtain a 500 µM stock solution Streptavidin-XL665 1 vial of 3 mgLyophilized  Reconstitute with distilled water (refer to product label) to obtain a 16.67 µM stock solution in streptavidin TK Antibody-Cryptate 1 vialLyophilized  Reconstitute with 1 mL of distilled water (refer to product label) to obtain the TK Antibody- Cryptate stock solution * Supplement Enzymatic buffer (SEB reagent) 1 vial Lyophilized  Reconstitute with 5 mL of distilled water to obtain a 2,500 nM SEB stock solution. 5x Enzymatic buffer HEPES 250 mM (pH7.0), NaN3 0.1%, BSA 0.05%, Orthovanadate 0.5 mM 1 vial of 50 mL Liquid 5x HTRF® Detection buffer HEPES 50 mM (pH7.0)with additives 1 vial of 200 mL Liquid 1x Storage: All kit components must be stored at +4°C until the expiration date printed on the product label. After reconstitution, the stock solutions can be stored 1 week at +4°C or dispensed into single use aliquots and stored at -20°C. Avoid repeated freezing and thawing. *Cryptate conjugate working solution (prepared with frozen stock solution) should be filtered before use to improve assay reproducibility. 3. Additional material required (not provided) Recommended Supplier* Stock solution to prepare Kinase Upstate (www.upstate.com) Follow supplier’s instructions ATP Sigma # A7699 5 mM in HEPES buffer 50 mM Enzymatic buffer supplements: The enzymatic buffer must be supplemented with any components required by the kinase of interest (See Appendix for further details). Recommended Supplier* Stock solution to prepare DTT Sigma # D0632 100 mM in distilled water MgCl2 Sigma # M1028 1 M (ready to use) MnCl2 Sigma # M1787 1 M (ready to use) * Suppliers’ names are indicative. Storage: Stock solutions should be aliquoted and stored at -80°C for kinases and -20°C for ATP and DTT. 3 4. Assay protocol for 384w low volume plate (20µL) Add assay components (working solutions) in the following order: 5 µL Sa-XL665 in EDTA + 5 µL TK Antibody-Eu(K) in EDTA 4 µL Compounds (or kinase buffer)* + 2 µL TK Substrate-biotin + 2 µL Kinase + 2 µL ATP or 37°C The kinase reaction is started by the addition of ATP (step 1) and is stopped by the addition of the detection reagents which contain EDTA (step 2). The incubation period for the enzymatic step is optimized depending on the kinase (§ 8.3). For a 384w low volume plate, we recommend the 10 µL enzymatic step and the 10 µL detection step for a final assay volume of 20 µL. For a 96 half well plate (100 µL), each addition volume is simply multiplied by 5. * For low volume compound addition, adjust volume to 4 µL with 1x kinase buffer. Keep DMSO ≤ 2% in the enzymatic step. 5. Preparation of the working solutions The working solutions are prepared from stock solutions (§ 2-3) by following the instructions below: Buffer to prepare Kinase buffer 1X Prepare “enzymatic buffer 1X “ by diluting 1 volume of enzymatic buffer 5X with 4 volumes of distilled water and any supplements required by the kinase of interest, i.e. DTT, MgCl2,MnCl2, etc. (see appendix). For some tyrosine kinases , the addition of the SEB reagent in the kinase buffer 1X will help to catalyze the enzymatic reaction (refer to §8.1 : SEB titration). HTRF® Detection buffer Ready to use Component working solutions to prepare Compounds Dilute compound stock solution with kinase buffer to prepare a working solution which has 2.5X the required final concentration for the enzymatic step TK Substrate-biotin Dilute the substrate stock solution (500 µM) with kinase buffer to prepare a working solution which has 5X the required final concentration for the enzymatic step Kinase Dilute the kinase stock solution with kinase buffer to prepare a working solution which has 5X the required final concentration for the enzymatic step ATP* Dilute the ATP stock solution (5 mM) with kinase buffer to prepare a working solution which has 5X the required final concentration for the enzymatic step Sa-XL665** Dilute the SaXL665 stock solution (16.67 µM) with HTRF® detection buffer to prepare a working solution which has 4X the required final concentration for the assay (20 µL) TK-Antibody-Cryptate Dilute the TK Antibody-Cryptate stock solution 1:100 in HTRF® detection buffer to obtain the working solution (e.g. for 20,000 tests :1 mL of TK Antibody-Cryptate stock solution + 99 mL of HTRF® detection buffer) * for an ATP 100 µM in the enzymatic step, prepare a 500 µM ATP working solution. ** for an Sa-XL665 125 nM final concentration, prepare a 500 nM SaXL665 working solution. 4 Precautions: - It is recommended to prepare the required amount of Kinase buffer 1X (supplemented with ions , DTT required by the enzyme of interest and SEB) just before use as DTT and SEB are stable only one day at 2-8°C once diluted in the enzymatic buffer . - Working solutions cannot be stored and must be used immediately. - The enzyme working solution must be kept in an ice bath for the time of the experiment (to avoid degradation). - HTRF® cryptate conjugate concentration have been set for optimal assay performances. Note that any dilution or improper use of the detection reagents will impair the assay’s quality. 6. Kinase assay and controls The kinase assay is performed as described below, using three different controls: Negative control: used to calculate the specific signal. Appropriate Negative controls must be prepared for each Sa-XL665 concentration tested (§ 8.4). Buffer control: used to make sure that buffers are not contaminated by Cryptate and do not generate any background fluorescence. Cryptate control: used to check the Cryptate signal at 620 nm. Kinase assay Controls Enzymatic step (10 µL) Sample Negative Cryptate Buffer Compounds (or kinase buffer) 4 µL 4 µL kinase buffer 10 µL kinase buffer 10 µL kinase buffer TK Substrate-biotin 2 µL 2 µL Kinase 2 µL 2 µL kinase buffer ATP 2 µL 2 µL Seal plate and incubate at RT or 37°C Detection step (10 µL) Sa-XL665 5 µL 5 µL 5 µL detection buffer 10 µL detection buffer TK Ab-Cryptate 5 µL 5 µL 5 µL Seal plate and incubate 1h at RT Remove plate sealer and read on an HTRF® compatible reader* *More information at http://www.htrf.com/technology/htrfmeasurement/compatible_readers/ Data reduction: The fluorescence is measured at 620 nm (Cryptate) and 665 nm (XL665). A ratio is calculated (665/620) for each well. Results are expressed as follows: Specific signal = Ratio (Sample) – Ratio (Negative control) Ratio = (665 nm/620 nm) x 104 Mean ratio= S ratio/2 (n=2) CV%= (Std deviation/Mean ratio)*100 5 7. Assay miniaturization and flexibility When used as suggested, the kit will provide sufficient reagents for 20,000 tests using a 384-well low volume plate in 20 µL final assay volume (HTRF® packaged basis): 5 µL Sa-XL665 in EDTA + 5 µL TK Antibody-Eu(K) in EDTA 4 µL Compounds (or kinase buffer) + 2 µL TK Substrate-biotin + 2 µL Kinase + 2 µL ATP or 37°C Other plate formats (96-half-well or 1536-well) and final volumes (100 µL to less than 10 µL) can be used by simply proportionally adjusting each addition volume in order to maintain the concentrations as for the 20 µL final assay volume. Miniaturization Assay format: 1536-well (10 µL) 384-well low volume (20 µL) 96 half-well (100 µL) Compounds/kinase/ Substrate/ATP 2 / 1 / 1 / 1 µL 4 / 2 / 2 / 2 µL 20 / 10 / 10 / 10 µL Sa-XL665 2.5 µL 5 µL 25 µL TK-Ab-Cryptate 2.5 µL 5 µL 25 µL Number of test per kit 40,000 tests 20,000 tests 4,000 tests Plate references: 96 half-well plate (Costar # 3694 or equivalent), 384-well low volume plate (Greiner # 784076), 1536-well (Greiner # 782086). 8. Optimization of the kinase assay A typical development for an HTRF® KinEASE™-TK assay consists of the following steps: 1. SEB titration 2. Enzyme titration 3. Kinetic study 4. Substrate titration 5. ATP titration 6. Biotin/streptavidin ratio optimization 7. Inhibitor IC50 determination Final concentrations of the assay components used for kinase assay optimization are : Conc. Max. Conc. Min. TK-Substrate-biotin Final conc. in the enzymatic step (10 µL) 2 µM 0.97 nM Kinase 10 ng/well (1 ng/µL) 0.1 ng/well (0.01 ng/µL) ATP 300 µM 1.7 nM Sa-XL665 Final conc. in the final assay volume (20 µL) 125 nM 0.06 nM TK-Ab-Cryptate Ready to use Ready to use 6 8.1. SEB titration Some tyrosine kinases may require the addition of SEB reagent in the kinase buffer 1x for optimal enzymatic activity. This step enables the optimal SEB concentration, i.e. that for which the signal reaches 80% of the maximum, to be determined. Prepare a series of Kinase buffer 1X supplemented with different concentrations of SEB ranging from 125 nM to 0 nM (Control kinase buffer 1X): dilute SEB stock solution 2500 nM 1/20 in Kinase buffer 1X to get a SEB working concentration of 125 nM . Next, make 2 fold serial dilutions in kinase buffer1x to reach a SEB working concentration of 2nM. The different SEB supplemented kinase buffers are dispensed under 4µL (first dispensing step - refer to assay protocol § 4). To calculate SEB final concentration in the enzymatic step, divide the SEB working concentrations by 2.5. The vial of SEB reagent enables to perform 1,000 tests using a final SEB concentration of 50 nM during the enzymatic step (maximal SEB concentration required on a selection of 59 tyrosine kinases - see Appendix for further details). Kinase , TK-substrate-biotin and ATP must be diluted in kinase buffer 1X non supplemented with SEB. For this step, we recommend the use of a fixed concentration of kinase (10 ng/well * in 384 half well plates, 20µL final volume) and saturating concentrations of TK-substrate-biotin (1 µM)* and ATP (100 µM)*. Allow the enzymatic reaction to run for 30 mn at RT. Add the detection reagents. The biotin/streptavidin molar ratio must be 8/1 (i.e. 62.5 nM Sa-XL665**). The signal (ratio sample (with enzyme) – ratio negative) is plotted versus the different SEB concentrations. Determine the optimal SEB concentration for the following experiments, targeting the SEB concentration for which the signal reaches 80% of the maximum. NB: For the next experiments, prepare the kinase buffer 1X supplemented with SEB at the desired working concentration just before use. This buffer is stable one day at 2-8°C. Enzyme, substrate, ATP and compound can be diluted directly in the SEB kinase buffer. * Final concentrations in the enzymatic step (10 µL). ** Final assay concentration (20 µL). 8.2. Enzyme titration This step allows the optimal enzyme concentration (for which the signal reaches 80% of the maximum) to be determined. A compromise may be found between a high assay signal and the enzyme consumption. For this step, a fixed concentration of the TK-substrate-biotin (1 µM) and ATP (100 µM) should be tested with the following enzyme concentrations 10; 2; 1; 0.1 ng/well. Allow the enzymatic reaction to run for 30 mn. The biotin/streptavidin ratio of 8/1 must be used (i.e. 62.5 nM Sa-XL665). 8.3. Kinetic study Enzyme kinetic depends on the kinase and substrate concentrations. A time course study is performed using a constant concentration of kinase (determined in the previous experiment), ATP (100 µM) and substrate (1 µM). The reaction is stopped at different end points by the addition of the detection reagents (1, 2, 5, 10, 15, 30, 60 min). The biotin /streptavidin ratio must remain constant and equal to 8/1 (i.e. 62.5 nM Sa-XL665). The signal is then plotted versus the different end points. Determine the linear part of the time course (correlation coefficient R2 > 0.99) and from this section, the optimal incubation time to use for the next experiments. 8.4. Substrate titration This step allows the determination of substrate Km (app). Use the optimal enzyme concentration (§ 8.2) and a saturating ATP concentration (100 µM). We recommend testing different TK substrate-biotin concentrations ranging from 2 µM to 0.97 nM (two fold serial dilutions). The kinase reaction is stopped at the previously determined optimal incubation period. During the detection step, it will be necessary to adjust the concentration of the SA-XL665 for each TK substrate-biotin concentration, in order to keep the biotin/streptavidin ratio constant at 8/1 as described in the following table. Furthermore, since the background may rise with increasing XL665 concentrations, it is necessary to run a negative control (no enzyme) for each Sa-XL665 concentration. 7 TK Substrate-biotin Sa-XL665 Final conc. in the enzymatic step (10 µL) Final assay conc. (20 µL) Final assay conc. (20 µL) 2 µM 1 µM 0.125 µM 1 µM 0.5 µM 62.50 nM 0.5 µM 0.25 µM 31.25 nM 0.25 µM 0.125 µM 15.61 nM 0.125 µM 62.50 nM 7.81 nM 62.50 nM 31.25 nM 3.90 nM 31.25 nM 15.61 nM 1.95 nM 15.61 nM 7.81 nM 0.97 nM 7.81 nM 3.90 nM 0.48 nM 3.90 nM 1.95 nM 0.24 nM 1.95 nM 0.97 nM 0.12 nM 0.97 nM 0.485 nM 0.06 nM The plot of the specific signal (ratio sample (with enzyme) – ratio negative) versus the substrate concentrations is then fitted to Michaelis-Menten or Lineweaver-Burke equations to calculate the substrate Km (app). 8.5. ATP titration This step allows the determination of ATP Km (app). Use the optimal enzyme concentration and a saturating TK-substrate-biotin concentration (1 µM). We recommend testing ATP concentrations ranging from 300 µM to 1.7 nM (three fold serial dilutions). The kinase reaction is stopped at the optimal incubation period by adding the detection reagents. During the detection step, the biotin/streptavidin ratio must be fixed at 8/1 (62.5 nM SA-XL665). As in the previous step, the Km (app) value must be determined from this experiment using either a Michaelis-Menten or a Lineweaver-Burke plot. 8.6. Biotin/streptavidin ratio optimization The optimization of the biotin/streptavidin ratio is an important step which may lead to a substantial increase in signal. Streptavidin-XL665 solutions are prepared in order to cover 2/1, 4/1, 8/1 biotin/streptavidin ratios. The test is run using the optimal enzyme, ATP and substrate concentrations (§ 8.1-5). Negative controls corresponding to each Sa-XL665 concentration must be used as this reagent has a direct contribution to the background level. 8.7. Inhibitor IC50 determination The kinase activity is tested over a broad range of inhibitor concentrations to generate a dose response curve. The test is generally run using the previously determined optimal assay conditions. 9. HTRF® KinEASE™ product line The most appropriate HTRF® KinEASE™or HTRF® KinEASE™-TK assay system can be used depending on your specific applications (see table below). HTRF® KinEASE™ kits consist of substrate(s)-biotin, antibody labeled with Europium Cryptate (Eu(K)), Sa-XL665, enzymatic and HTRF® detection buffers. Three packaging sizes are available using a 20 µL test format. The kit discovery that includes the three STK substrates-biotin (1, 2 and 3) is designed to quickly test the desired Ser/Thr kinase. Once the substrate that works with the desired kinase has been identified, the kit S1, S2 or S3 including the most appropriate substrate can be used for kinase assay development. If larger volumes are required for HTS or profiling, kits are available in Bulk or Jumbo sizes. The kit reagents like substrate-biotin, Sa-XL665 and assay buffers can also be ordered separately. Copyright © 2009 CIS bio international, France - KinEASE™ is a trademark of Millipore. HTRF®, TRACE®, and the HTRF™ logo are trademarks belonging to CIS bio international. HTRF® products are manufactured under one or more of the following patents and foreign equivalent : EP 0 180 492 / US 4,927,923 / US 5,220,012 / US 5,432,101 – EP 0 321 353 / US 5,457,185 / US 5,534,622 / US 5,346,996 / US 5,162,508 – EP 0 539 477 / US 5,512,493 – EP 0 539 435 / US 5,627,074 – EP 0 569 496 / US 5,527,684. CIS bio international hereby grants to those buying HTRF® products from CIS bio international or its affiliates / distributors, a worldwide, non-exclusive, royalty-free, limited license to use HTRF® technology with said products for in-house life science research only. Signal amplification and correction using HTRF® technology are covered by the following U.S. patents or patent applications and foreign equivalents : US 5,512,493 – US 5,527,684 – US 6,352,672. HTRF® KinEASE™ for Serine / Threonine kinases Description Quantity Cat no. HTRF® KinEASE™-STK discovery (STK substrates 1, 2 and 3-biotin ) 1,000 tests 62ST0PEB HTRF® KinEASE™-STK S1 (STK substrate 1-biotin) 1,000 tests Bulk 20,000 tests Jumbo 100,000 tests 62ST1PEB 62ST1PEC 62ST1PEJ HTRF® KinEASE™-STK S2 (STK substrate 2-biotin) 1,000 tests Bulk 20,000 tests Jumbo 100,000 tests 62ST2PEB 62ST2PEC 62ST2PEJ HTRF® KinEASE™-STK S3 (STK substrate 3-biotin) 1,000 tests Bulk 20,000 tests Jumbo 100,000 tests 62ST3PEB 62ST3PEC 62ST3PEJ STK substrate 1-biotin 50 µg/vial500 µg/vial 61ST1BLE 61ST1BLC STK substrate 2-biotin 50 µg/vial500 µg/vial 61ST2BLE 61ST2BLC STK substrate 3-biotin 50 µg/vial500 µg/vial 61ST3BLE 61ST3BLC HTRF® KinEASE™ for Tyrosine kinases Description Quantity Cat no. HTRF® KinEASE™-TK 1,000 tests Bulk 20,000 tests Jumbo 100,000 tests 62TK0PEB 62TK0PEC 62TK0PEJ TK substrate -biotin 50 µg/vial500 µg/vial 61TK0BLE 61TK0BLC Companion products Description Quantity Cat no. Sa-XL665 250 µg 1 mg 3 mg 610SAXLA 610SAXLB 610SAXLG 5x Enzymatic buffer HTRF® Detection buffer 50 mL 200 mL 62EZBFDD 62SDBRDF