TECHNICAL DATA SHEET AlphaLISA® Research Reagents Caution: For Laboratory Use. A research chemical for research purposes only Anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) Acceptor Beads Product No.: AL121C (250 µg) AL121M (5 mg) AL121R (25 mg) Lot No.: 2010404 Product Formats Catalog # Size Volume Assay points* AL121C AL121M AL121R 250 µg 5 mg 25 mg 0.05 mL 1 mL 5 mL 500 10 000 50 000 *The number of assay points is based on an assay volume of 25 µL using a final bead concentration of 20 µg/mL in 384-well format. Manufacturing Date: March 20, 2015 Product Information Description: Anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) AlphaLISA Acceptor beads at 5 mg/mL in PBS pH 7.2, supplemented with 0.05% Proclin-300 as a preservative. Source of the antibody: rabbit monoclonal. Application: This product is designed to detect human histone H3 di- and mono-methylated at lysine 27 ® (H3K27me2-1) in a homogeneous AlphaLISA assay. Broad species cross-reactivity is expected based on sequence similarity. Storage: Store in the dark at 4˚C. Stability: This product is stable for at least 12 months from the manufacturing date when stored in its original packaging at the recommended storage conditions. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. Quality Control ® Lot-to-lot consistency is confirmed by a Quality Control AlphaLISA titration assay read on an EnVision Alpha HTS instrument. Maximum signal and EC50 value are determined using the biotin-H3K27me2 peptide. Minimum signal is derived from the non-modified biotin-H3 (21-44) peptide at the concentration giving the specified maximum signal. We certify that this product meets our quality release criteria. Maximum counts may vary between lots and depending on assay conditions. Maximum signal: Minimum signal: EC50: TDS-AL121-03 Page 1 of 5 855673 counts 254 counts 19.2 nM AlphaLISA Signal (counts) Typical Data 600,000 biotin-H3 (21-44) 500,000 biotin-H3K27me1 biotin-H3K27me2 biotin-H3K27me3 400,000 300,000 biotin-H3K36me1 biotin-H3K36me2 biotin-H3K36me3 200,000 100,000 0 - -10 -9 -8 -7 -6 -5 Log [biotin-peptide] (M) Specificity of anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) Acceptor Beads. Histone H3-derived peptides with different epigenetic marks were titrated in QC buffer (50 mM Tris-HCl pH 8.0). Signal was detected with an EnVision Alpha HTS instrument 2102. The hook effect observed at higher peptide concentrations is typical of three-component assays and occurs when peptide concentrations exceed the binding capacity of the Alpha Streptavidin Donor and/or AlphaLISA Acceptor beads. Shifts in affinity might be observed in different enzymatic assay buffers. Technical notes presenting the optimization of an AlphaLISA EZH2 histone H3 lysine 27 methyltransferase assay and JMJD3 histone H3 lysine 27 demethylase assay using anti-H3K27me2-1 AlphaLISA Acceptor beads are available on our website at www.perkinelmer.com/epigenetics. Peptide Titration Assay Peptide titration provides a means to verify product performance. It is used as our Quality Control release test. The following reagents and materials are recommended: Item Supplier Catalog # AlphaScreen Streptavidin Donor Beads PerkinElmer 6760002S (1 mg) 6760002 (5 mg) 6760002B (50 mg) Histone H3K27me2 peptide, biotinylated AnaSpec 64366 Histone H3 (21-44) peptide, biotinylated AnaSpec 64440 AlphaLISA 5X Epigenetics Buffer 1 Kit* PerkinElmer AL008C (10 mL Kit) AL008F (100 mL Kit) White opaque OptiPlate™-384 PerkinElmer 6007290 TopSeal™-A Adhesive Sealing Film PerkinElmer 6050195 PerkinElmer - ® ® ® ® EnSpire or EnVision Multilabel Plate Reader with Alpha option TDS-AL121-03 Page 2 of 5 *The two-component AlphaLISA 5X Epigenetics Buffer 1 Kit includes AlphaLISA 5X Epigenetics Buffer 1 and AlphaLISA 30X Buffer Supplement. The 1X AlphaLISA Epigenetics Buffer 1 may appear cloudy. However, this will not affect assay performance. The 1X Epigenetics Buffer 1 should be used within 16 hours. These microplates can also be used with this product: Item Recommended Assay Volume Supplier Catalog # White opaque OptiPlate-96 100 µL PerkinElmer 6005290 White ½ AreaPlate-96 50 µL PerkinElmer 6005560 Light gray AlphaPlate™-384 25 µL PerkinElmer 6005350 ProxiPlate™-384 Plus 12.5 µL PerkinElmer 6008280 Light gray AlphaPlate-1536 5 - 10 µL PerkinElmer 6004350 Recommendations The volume indicated on each tube is guaranteed for single pipetting. Multiple pipetting of the reagents may reduce the theoretical amount left in the tube. To minimize loss when pipetting beads, it is preferable not to prewet the tip. AlphaScreen Donor beads are light-sensitive. All Alpha assays using the Donor beads should be performed under subdued laboratory lighting (< 100 lux). Green filters (LEE 090 filters (preferred) or Roscolux filters #389 from Rosco) can be applied to light fixtures. Sodium azide should not be added to the stock reagents. High concentrations of sodium azide (> 0.001 % final in the assay) might decrease the AlphaLISA signal. Centrifuge the tubes briefly before use to improve recovery of content (2,000 x g, 10-15 sec). Resuspend all reagents by vortexing before use. Use Milli-Q grade H2O (18 MΩ•cm) to prepare the 1X AlphaLISA Epigenetics Buffer 1. When diluting peptides, change tips after each dilution. When loading reagents in the assay microplate, change tips after each reagent addition and between each set of reagents. Small volumes may be prone to evaporation. It is recommended to cover microplates with a TopSeal-A Adhesive Sealing Film to reduce evaporation during incubation. Microplates are read with the TopSeal-A Film on the plate. Total signal varies with temperature and incubation time. For consistent results, identical incubation times and temperature should be used for all plates. The AlphaLISA signal is detected with an EnSpire or EnVision Multilabel Plate Reader equipped with the ALPHA option using the AlphaScreen standard settings (i.e. Total Measurement Time: 550 ms, Excitation Time: 180 ms, Mirror: D640as, Emission Filter: M570w, Center Wavelength 570 nm, Bandwidth 100 nm, Transmittance 75%). ® TDS-AL121-03 Page 3 of 5 Quality Control Protocol PLEASE READ RECOMMENDATIONS ON PAGE 3 BEFORE USE This titration protocol is designed for 12 dilutions of 2 peptides (biotin-H3K27me2 and non-modified biotin-H3 (21-44) peptides) with triplicate determinations. Peptide concentrations are indicated for a 10 µL volume. Final concentration of Alpha Donor and AlphaLISA Acceptor beads in the 25 µL total assay volume is 20 µg/mL. 1) Assay Buffer: The Assay Buffer used for biotin-peptide dilution is 50 mM Tris-HCl pH 8.0. 2) Serial dilutions of biotin-peptide in Assay Buffer: Prepare dilution series for each biotin-peptide as follows, changing tip for each dilution: Tube Volume of biotin-peptide Volume of Assay Buffer (µL) [biotin-peptide] (M) in 10 µL A B C D E F G H I J K L 4 µL of 500 µM stock 60 µL of tube A 60 µL of tube B 60 µL of tube C 60 µL of tube D 60 µL of tube E 60 µL of tube F 60 µL of tube G 60 µL of tube H 60 µL of tube I 60 µL of tube J 0 196 1.0E-5 3.0E-6 1.0E-6 3.0E-7 1.0E-7 3.0E-8 1.0E-8 3.0E-9 1.0E-9 3.0E-10 1.0E-10 0 140 120 140 120 140 120 140 120 140 120 100 3) Preparation of 1X AlphaLISA Epigenetics Buffer 1: Add 2.0 mL of AlphaLISA 5X Epigenetics Buffer 1 and 0.33 mL of AlphaLISA 30X Buffer Supplement to 7.67 mL H2O. The cloudy appearance of the buffer is normal. Use the 1X Epigenetics Buffer 1 within 16 hours. 4) Preparation of 5X AlphaLISA Acceptor Beads (100 µg/mL): Add 10 µL of 5 mg/mL AlphaLISA Acceptor beads to 490 µL of 1X AlphaLISA Epigenetics Buffer 1. 5) Preparation of 2.5X Streptavidin Donor Beads (50 µg/mL): Keep the beads under subdued laboratory lighting. Add 10 µL of 5 mg/mL Streptavidin Donor beads to 990 µL of 1X AlphaLISA Epigenetics Buffer 1. TDS-AL121-03 Page 4 of 5 6) In a white opaque OptiPlate-384 microplate: Add 10 µL of each biotin-peptide dilution Add 5 µL of 5X AlphaLISA Acceptor beads (20 µg/mL final) Incubate 60 min at 23˚C Add 10 µL of 2.5X Streptavidin Donor beads (20 µg/mL final) Incubate 30 min at 23˚C in the dark Read using EnSpire or EnVision Multilabel Plate Reader with Alpha option Please visit our website for additional information on the AlphaLISA technology at www.perkinelmer.com/AlphaTech. This product is not for resale or distribution except by authorized distributors. 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