08 Jan 15 Page 1 of 18 Z -LYTE Assay Setup Guide on the BMG LABTECH CLARIOstar Reader The BMG LABTECH CLARIOstar Microplate Readers were tested for compatibility with the Life Technologies Z -LYTE Assay using the Z'-LYTE Ser/Thr 11 Kit (PV3670) and FRAP1 (mtor) kinase (PV4753). The following document is intended to demonstrate setup of this instrument and provide representative data. For more detailed information and technical support of Life Technologies assays please call 1-800-955-6288 extension 40266. For more detailed information and technical support of BMG LABTECH instruments or software, please contact BMG LABTECH at 1-877-264-5227 or www.bmglabtech.com. A. Recommended Optics wavelength (nm) Wavelength Selection Excitation 400-12 (or similar) Emission 1 445-12 (or similar) Emission 2 520-12 (or similar) LVF Monochromator LVF Monochromator LVF Monochromator Emission 1 Dichroic 422.5 Linear Variable Dichroic Emission 2 Dichroic 460 Linear Variable Dichroic
08 Jan 15 Page 2 of 18 B. Instrument Setup 1. Make certain plate reader is turned on, and open up CLARIOstar Control software on computer. Insert plate into plate reader. 2. Setup protocol for Z'-LYTE, select "Manage Protocols" from the Protocols tab in the ribbon style menu at the top of the window.
08 Jan 15 Page 3 of 18 3. At this point, a new screen will open (below). Click on the Show all test protocols or "Fluorescence Intensity" button on the left side of the screen, then select New from the tabs at the bottom.
08 Jan 15 Page 4 of 18 4. A new window will pop up. Select Fluorescence Intensity and Endpoint and then select OK.
08 Jan 15 Page 5 of 18 5. A new Protocol window will open automatically. Enter a test name, select plate type, type 2 into box beside No. of multichromatics and in Optic select Top optic. In Speed and Precision select Rapid or Precise or click on.
08 Jan 15 Page 6 of 18 6. Additional selections are now made available to change the speed at which the plate is read. When finished, select the "Layout" tab at the top of the Protocol window.
08 Jan 15 Page 7 of 18 7. Select the wells you wish to read. Note in this step you can select to designate blanks, positive controls, etc. but for this case we marked all wells "Sample" and calculations were performed manually. When finished, select the Multichromatic tab.
08 Jan 15 Page 8 of 18 8. Adjust the monochromator settings by typing in the desired wavelength bandpass. For both chromatics, type in 400-12 for the Excitation. For chromatic 1, type in an Emission of 445-12 and for chromatic 2, type in an Emission 520-12. The Dichroic settings will be adjusted automatically. At this time you can select Start measurement to run this protocol immediately or OK to save this protocol.
08 Jan 15 Page 9 of 18 9. If you selected OK in the previous step you will return to the initial settings window. Select the Start measurement button in the menu bar at the top of the screen.
08 Jan 15 Page 10 of 18 8. A new window will appear allowing you to select which of your test protocols to be run. Select the protocol created for Z'-LYTE, and then press OK.
08 Jan 15 Page 11 of 18 9. A new window will appear. Place the plate into the reader, and select a well to use for adjusting gain and focus by highlighting the well of your choice. The gain or sensitivity can be adjusted for the highlighted channel at this point; in this case a positive control (high FRET) well was used. When finished, click on the "Start Adjustment" tab. This will procedure will need to be followed for both channels.
08 Jan 15 Page 12 of 18 10. In a moment, the instrument will have calculated its optimal focal height and the gain adjustments necessary. When finished with adjusting both channels, click on the "Start Measurement" tab to read.
08 Jan 15 Page 13 of 18 11. When CLARIOstar has finished reading, collect the data by clicking the MARS icon in the Results portion of the ribbon on the toolbar at the top of the window. This will automatically redirect you to the MARS data evaluation file which collects run data. Select the run of interest from the list to open, and view data in a plate layout format.
08 Jan 15 Page 14 of 18 C. Z'-LYTE Kinase Assay using FRAP1 (mtor) Purpose The following is a sample assay performed for demonstration purposes. The section below describes how the data was obtained, and is not intended for use as an assay protocol. We recommend all first-time users follow the appropriate protocols and/or validation packets provided with their specific assay kits, and include all proper controls. The instrument settings above would be sufficient for any Z'-LYTE assay and were optimized for high throughput. The information below is provided as representative data. Assay was run at ATP Km apparent and a kinase concentration producing approximately 30-40% of maximal phosphorylation, as discussed in the Z'-LYTE protocols. ATP and kinase concentrations should be optimized for each kinase by the actual user. Specific Z -LYTE assay protocols and setup information from Life Technologies own in-house SelectScreen Custom Profiling Z -LYTE -based kinase assay service can be located at the following link: http://www.lifetechnologies.com/selectscreen and select the SelectScreen Kinase Profiling Service and select Technology Overview. At a Glance Step 1: Step 2: Step 3: Step 4: Setup the instrument and plate layout. Dilution series of inhibitor. Prepare assay plate with controls. Read. Materials Required Component Part Number Storage FRAP1 (mtpor) kinase PV4753-80 C Z'-LYTE -LYTE Assay Kit Ser/Thr 11 containing PV3670 Z'-LYTE Kinase Assay Kit - Ser/Thr 11 Peptide PV3671-20 C Z'-LYTE Ser/Thr 11 Phospho-Peptide PV3672-20 C 5X Kinase Buffer A PV3189 20-30 C ATP PV3227-20 C Development Reagent B PV4126-80 C Development Buffer P3127 20-30 C FRAP1 (mtor) Kinase Buffer User prepared see http://www.lifetechnologies.com/selectscreen
08 Jan 15 Page 15 of 18 Materials Required But Not Supplied 384-well assay plates. We recommend black Corning 384-well, low-volume, round-bottom (non-binding surface) assay plates (Corning #4514). Other blackwalled, low-binding assay plates, while not tested, may be suitable. Multi-channel pipettor (12 channels) or any pipetting device that can accurately deliver repeated volumes of 2.5 µl and 5 µl. 96-well microplates that can accommodate 300 µl per well PI-103 (CAS 371935-74-9), MW 348.4 DMSO Procedure 1. Prepare initial 100X serial dilution curves in Row A of a 96-well plate: Dilute PI-103 to a 100X initial concentration in 100% DMSO (500 µm). Prepare a set of 3-fold serial dilutions from the initial concentration in a 96-well plate: Add 30 µl DMSO in wells A2 - A10. Add 45 µl of 500 µm of PI-103 in A1. Add 15 µl from well A1 to well A2, and then mix well A2, and take 15 µl from well A2 and add to well A3, mix, and so on. Figure 1: Schematic of initial compound dilution. PI-103 was titrated from a 500 µm starting concentration in the initial dilution series by preparing a 3-fold dilution in 100% DMSO. A secondary dilution to 4X was then prepared in the rows below the initial dilution curve (lighter gray) using kinase buffer. 2. Prepare a 4X Intermediate Dilution of PI-103 in Row B: Add 48 µl of 1X Kinase Buffer A into all wells B1 B10. Add 2 µl of diluted inhibitor from the wells in
08 Jan 15 Page 16 of 18 Row A to wells in Row B. This will produce a final serial dilution starting at 20 µm (B1), which will then produce a final assay concentration starting at 5 µm. 3. Prepare an Assay Plate: Prepare duplicate wells. Add 2.5 μl of the 4X compound dilutions per well into a low volume NBS, 384-well plate (Corning Cat. # 4514)., Using row 1, Columns 1 and 2 are replicates of the highest concentration, Columns 3 and 4 are replicates of the second highest concentration, etc. to column 20. 4. Add 2.5 μl of 1X Kinase Buffer A alone to Columns 21 and 22 (0% inhibition, no compound control), 23 (0% phosphorylation control, no kinase added) and 24 (Phosphopeptide 100% phosphorylation positive control) 5. Add 5 μl of the 2X Peptide/Kinase Mixture (prepared in mtor kinase buffer) to Row 1 Columns 1-22. DO NOT ADD TO COLUMN 23 OR 24. 0% phosphorylation control: Add 5 μl of 2 μm substrate alone without kinase to column 23, rows A-L. 100% phosphorylation control: Add 5 µl of 2 µm phosphopeptide control substrate to column 24, rows A-L. Buffer-only reference: Add 5 µl Kinase Buffer A alone to the remaining 8 wells (columns 23 and 24, rows M-P). 6. Add 2.5 μl of 4X ATP Solution (200 μm) per well to all columns to start reaction. 7. Shake assay plate on a plate shaker for 30 seconds. 8. Incubate assay plate for 60 minutes at room temperature. 9. Add 5 μl of the Development Reagent Solution to each well. Use the lot-specific dilutions indicated on your Certificate of Analysis as dilutions may vary based upon Z'-LYTE peptide and Development Reagent A lot. 10. Shake plate again on a plate shaker for 30 seconds. 11. Incubate for 60 minutes at room temperature. 12. Read and analyze as directed in the protocol.
08 Jan 15 Page 17 of 18 A B C D E F G H I J K L M N O P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Figure 2: Assay Plate Schematic. Compound titrations, duplicate 10-point titration shown in row A columns 1-20 with known inhibitor (e.g. PI-103). Columns 21 and 22 prepared without any inhibitor as kinase activity controls (purple), column 23 prepared with no kinase, 0% phosphorylation, (blue) and column 24 prepared using phosphopeptide control, 100% phosphorylation, (green). Note 8 wells in bottom right (yellow M23 P24), were prepared without any inhibitor or substrates, as buffer controls. White wells can be used for the assay test compounds.
Emission Ratio Z -LYTE Compatible Microplate Reader 08 Jan 15 Page 18 of 18 D. Results 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 PI-103 IC50 = 49 nm 3.0 0.1 1 10 100 1000 10000 [PI-103] (nm) Figure 1: Z'-LYTE Kinase Assay. Z -LYTE Assay using Ser/Thr 11 (PV3670) with FRAP1 (mtor) (PV4753) performed using the BMG LABTECH CLARIOstar.