TECHNICAL DATA SHEET LANCE® Ultra Research Reagents Caution: For Laboratory Use. A product for research purposes only Eu-W1024-labeled Anti-phospho-Histone H3 (Ser10) Antibody Product No.: TRF0210-D / TRF0210-M Lot No.: 1606201 Material Provided Format: TRF0210-D 10 µg (1 562 assay points*) TRF0210-M 100 µg (15 625 assay points*) *Assuming 40 fmol/ assay point Volume: 100 µL (TRF0210-D) or 1 mL (TFR0210-M) Manufacturing Date: March 12, 2012 Product Information Antibody: Europium-labeled mouse monoclonal antibody recognizing phospho-Ser10 in human Histone H3. Molecular Weight: 160 000 Storage Buffer: 50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.1% BSA and 0.05% sodium azide as preservative Stability: This product is stable for at least 12 months from the manufacturing date when stored in its original packaging and the recommended storage conditions. Storage Conditions: Store at 4°C Safety Note: The storage buffer contains sodium azide (NaN3) as a preservative. Disposal of all waste should be in accordance with local regulations. Quality Control The QC release specifications are based on spectrophotometric analysis of the labeled antibody. We certify that these results meet our quality release criteria. Labeling Ratio: Concentration: TDS-TRF0210-03 Page 1 of 3 6.9 (Eu/Ab) 100 µg/mL (0.625 µM) Recommended Assay Conditions AURORA B kinase: ATP titration Reagent Preparation: Prepare 1X Kinase Assay Buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20. Prepare a 2X Aurora B solution: dilute enzyme to a concentration of 0.2 nM in 1X Kinase Assay Buffer. Keep on ice. Prepare a 4X ULight-Histone H3 (Thr3/Ser10) Peptide solution: dilute ULight-Histone H3 (Thr3/Ser10) Peptide to a concentration of 200 nM in 1X Kinase Assay Buffer. Prepare a 4X ATP solution: dilute ATP to concentrations ranging from 40 nM to 4 mM (serial half-log dilutions) in 1X Kinase Assay Buffer. Keep on ice. Prepare 1X Detection Buffer: dilute 0.5 mL of 10X Detection Buffer with 4.5 mL of H2O. Prepare a 4X Stop solution*: dilute EDTA to a concentration of 40 mM in 1X Detection Buffer. Prepare a 4X Detection Mix: dilute Europium-anti-phospho-Histone H3 (Ser10) Antibody to a concentration of 8 nM in 1X Detection Buffer. Protocol: Pipet 5 µL of 2X Aurora B solution into a 384-well white OptiPlate-384 (0.1 nM final concentration). Add 2.5 µL of 4X ULight-Histone H3 (Thr3/Ser10) Peptide solution (50 nM final concentration). Add 2.5 µL of 4X ATP solution (10 nM to 1 mM final concentrations). Cover plate with TopSeal-A and incubate 60 min at 23°C. Add 5 µL of 4X Stop solution* and incubate 5 min at 23°C. Add 5 µL of 4X Detection Mix (2 nM Europium-anti-phospho-Histone H3 (Ser10) Antibody final concentration). Cover plate with TopSeal-A and incubate 60 min at 23°C. Remove TopSeal-A and read in TR-FRET mode at 665 nm (excitation at 320 or 340 nm). *Alternatively, the Stop solution and Detection Mix can be premixed and added together to the kinase reaction. TDS-TRF0210-03 Page 2 of 3 Typical ATP Titration Data LANCE Signal (665 nm) Aurora B kinase assay using ULight-Histone H3 (Thr3/Ser10) Peptide and Eu-anti-phospho-Histone H3 (Ser10) Antibody obtained using the EnVision® Multilabel Reader: 100000 75000 Km app = 1.8 M 50000 25000 0 - -9 -8 -7 -6 -5 -4 -3 -2 Log [ATP] (M) Suggested Materials Supplier Cat. No. Substrate: ULight™- Histone H3 (Thr3/Ser10) Peptide PerkinElmer TRF0125 Antibody: Eu- anti-phospho-Histone H3 (Ser10) Antibody PerkinElmer TRF0210 Kinase: Aurora B Carna Biosciences 05-102 Detection Buffer: LANCE Detection Buffer, 10X PerkinElmer CR97-100 Plate: OptiPlate™-384, white PerkinElmer 6007299 TopSeal™: TopSeal-A PerkinElmer 6050195 ® Please visit our website for additional resource: www.perkinelmer.com/LANCE This product is not for resale or distribution except by authorized distributors. LANCE® products may be covered by one or more the following patents: US 5,202,423 and US 5,324,825 LIMITED WARRANTY: PerkinElmer BioSignal Inc. warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and conform to specifications which accompany the product. 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