EPIGENETICS DRUG DISCOVERY SOLUTIONS Optimized for simplicity. Validated for your mark. Drug Discovery Solutions for Epigenetic and Other Post-Translational Modifications • Writers & Erasers of the Histone Code • p53 Acetylation & Deactylation Epigenetics, the study of changes in gene expression, is a rapidly growing field which presents a significant opportunity for drug discovery. As the understanding of the role of epigenetic changes in various disease states advances, the need for high quality reagents that can rapidly identify and optimize therapeutic molecules that target these enzymes is increasing. To address this need, PerkinElmer has developed first-in-class research tools built upon our homogeneous assay platforms, LANCE® Ultra and AlphaLISA® technologies. These platforms offer a combination of specificity, sensitivity, and throughput that other assay methods lack, all completely validated specifically for drug discovery research applications. Choose the platform that fits your workflow: Cellular Detection Kits In Vitro Tool Box AlphaLISA & Cell-Histone Buffers rapid and direct detection of endogenous histone modulation in a one-well, no-wash assay format LANCE Ultra – robust, ultra highthroughput TR-FRET technology AlphaLISA – high sensitivity, high throughput and ultimate flexibility - assay short peptides to full-length proteins PerkinElmer’s suite of epigenetic research reagents offers a faster, more efficient path in the race to identify promising epigenetic drug candidates: • Flexibility: Work with peptide or full-length protein substrates, or endogenous histones in cell-based format • Sensitivity: Robust and reproducible detection, even at low enzyme concentrations • Specificity: Reagents highly specific for the mark of interest • Simplicity: One-well, no-wash assay platforms amenable to automation and HTS • Validated: Detailed, ready-to-use protocols for over 30 epigenetic enzyme assays Cellular Detection Kits The AlphaLISA Epigenetic Cellular Detection Kits enable rapid and direct detection of endogenous modification of epigenetic marks on histone H3. These no-wash, all-in-one well assays are optimized for simplicity and throughput. • Simple assay protocol amenable to automation, eliminating tedious ELISAs and Westerns • Suitable for endogenous and recombinant cell lines, providing the flexibility to work with relevant cell models Assay Overview The AlphaLISA assay is based on a 3-step assay: cell preparation, extraction and detection* *Product format, protocol and detection principle for phospho marks is slightly different – see www.perkinelmer.com/surefire The detection is based on the immunocapture of the modified histones on the AlphaLISA beads ALL-IN-ONE WELL PROTOCOL PREPARATION Overnight EXTRACTION 25 min DETECTION 90 min Read Plate on EnVision® or EnSpire® Seed cells in plate 4 hours Add Cell-Histone Lysis Buffer 15 min Add Acceptor Beads and Biotinylated Anti-H3 60 min Treat cells – overnight Add Cell-Histone Extraction Buffer 10 min Add Donor Beads 30 min Cellular Detection Ordering and Assay Information Mark Detected Part Number Assay Points Methyl Modifications Part Number Assay Points Normalization Assay H3K4me2 AL716C500 AL716F5,000 H3K27me3 AL722C500 AL722F5,000 Acetyl Modifications H3K9ac AL714C500 AL714F5,000 H3K27ac AL720C500 AL720F5,000 Phospho Modifications H3Ser10p* Mark Detected TGRH3S500500 TGRH3S10K10,000 TGRH3S50K50,000 AlphaScreen SureFire® GAPDH Assay Kit* TGRGD500 500 TGRGD10K 10,000 TGRGDS50K50,000 Additional Items AlphaScreen® General IgG (Protein A) Detection Kit 6760617C 500 6760617M 10,000 6760617R50,000 * Requires AlphaScreen General IgG (Protein A) Detection Kit In Vitro Assay Toolbox The in vitro toolbox is comprised of validated off-the-shelf reagents for the detection of post-translational modifications of histone H3 and p53. Validated Enzyme Assays for Histone H3 Full-length protein assay available in Alpha SET7/9 Writers methylation Cellular assay available in Alpha demethylation Aurora B acetylation P300 HASPIN PRMT1 PRMT6 deacetylation P300 G9a phosporylation EZH2 NH2 – A R2T3 K4 Q TA R K9 S10 T G G K A P R K Q L AT K A A R K 27 S A PAT G G V K36K P H R . . . Erasers JMJD1A JMJD3 LSD1 JMJD2A HDAC1 SIRT1 SIRT2 SIRT3 HDAC1 HDAC3/NCOR2 HDAC2 HDAC6 HDAC10 HDAC3/NCOR1 SIRT1 SIRT2 SIRT3 SIRT7 HDAC1 HDAC2 HDAC3/NCOR1 HDAC3/NCOR2 HDAC6 HDAC10 CBP/p300 N – p53 – K382Ac SIRT1 K B + Enzyme SAM AlphaLISA and LANCE Ultra reagents to measure modifications on histone H3 and p53 SAH Me All detection reagents have been well characterized for their specificity at detecting the modification of interest, and validated for use K B with the specific enzymes depicted in the figure above. + Eu-Ab + ULight-SA AlphaLISA Beads LANCE Ultra TR-FRET FRET Eu Em 665 nm Emission 615 nm Excitation 320 or 340 nm Me ULight B Me K K Excitation 680 nm B With PerkinElmer, you can count on consistent performance that works every time. Plus we are continuing to expand our offering of epigenetic tools. If the product you need is not listed please inquire. In Vitro Ordering and Assay Information Acetyl Modifications Validated Assays Mark Detected Acetyltransferase Deacetylase H3K4 SIRT1 unmodified H3K9/K27 See Note 1 unmodified H3K9ac p300 H3K27ac p300 HDAC1 Alpha Technology LANCE Ultra (TR-FRET) AlphaLISA anti-mark Acceptor Beads LANCE Ultra Europium-labeled anti-mark Antibody (requires Alpha Streptavidin Donor beads) (requires LANCE Ultra ULight- Streptavidin) Part Number Quantity Assay Points Part Number Quantity Assay Points AL119C 250 µg 500 TRF0404-D 10 µg 1,500 AL119M 5 mg 10,000 TRF0404-M 100 µg 15,625 AL119R 25 mg 50,000 AL138C 250 μg 500 TRF0411-D 10 μg 1,500 AL138M 5 mg 10,000 TRF0411-M 100 μg 15,625 AL138R 25 mg 50,000 AL114C 250 µg 500 TRF0400-D 10 µg 1,500 AL114M 5 mg 10,000 TRF0400-M 100 µg 15,625 AL114R 25 mg 50,000 AL120C 250 µg 500 TRF0405-D 10 µg 1,500 AL120M 5 mg 10,000 TRF0405-M 100 µg 15,625 AL120R 25 mg 50,000 Non-Histone Target p53 K382ac p300 SIRT1 AL124C AL124M AL124R 250 µg 5 mg 25 mg 500 10,000 50,000 TRF0409-D TRF0409-M Note 1: For a detailed list of Validated Deacetylase Assays see the “Validated Enzyme Assays for Histone H3” figure above. 10 µg 100 µg 1,500 15,625 Methyl Modifications Validated Assays Alpha Technology LANCE Ultra (TR-FRET) AlphaLISA anti-mark Acceptor Beads LANCE Ultra Europium-labeled anti-mark Antibody Mark Detected Methyltransferase Demethylase (requires Alpha Streptavidin Donor beads) (requires LANCE Ultra ULight™-Streptavidin) Part Number Quantity Assay Points Part Number Quantity Assay Points H3R2me2 PRMT6 AL139C 250 µg 500 PRMT1 AL139M 5 mg 10,000 AL139R 25 mg 50,000 H3K4 LSD1 AL119C 250 µg 500 TRF0404-D 10 µg 1,500 unmodified AL119M 5 mg 10,000 TRF0404-M 100 µg 15,625 AL119R 25 mg 50,000 H3K4me1-2 SET7/9 AL116C 250 µg 500 TRF0402-D 10 µg 1,500 AL116M 5 mg 10,000 TRF0402-M 100 µg 15,625 AL116R 25 mg 50,000 H3K9/K27 JMJD1A AL138C 250 µg 500 TRF0411-D 10 µg 1,500 unmodified AL138M 5 mg 10,000 TRF0411-M 100 µg 15,625 AL138R 25 mg 50,000 H3K9me2 G9a JMJD2A AL117C 250 µg 500 TRF0403-D 10 µg 1,500 JMJD2C AL117M 5 mg 10,000 TRF0403-M 100 µg 15,625 AL117R 25 mg 50,000 H3K27me2-1 EZH2 JMJD3 AL121C 250 µg 500 TRF0406-D 10 µg 1,500 AL121M 5 mg 10,000 TRF0406-M 100 µg 15,625 AL121R 25 mg 50,000 H3K27me3 G9a AL122C 250 µg 500 TRF0407-D 10 µg 1,500 AL122M 5 mg 10,000 TRF0407-M 100 µg 15,625 AL122R 25 mg 50,000 H3K36me2 JMJD2A AL123C 250 µg 500 TRF0408-D 10 µg 1,500 AL123M 5 mg 10,000 TRF0408-M 100 µg 15,625 AL123R 25 mg 50,000 Phospho Modifications** Quantity/ Mark Detected Product Description Part Number Assay Points H3Thr3p LANCE Ultra Europium-labeled anti-phospho-Histone H3 (Thr3) Antibody TRF0211-D 10 µg / 1,500 LANCE Ultra Europium-labeled anti-phospho-Histone H3 (Ser10) Antibody H3Ser10p LANCE Ultra ULight-Histone H3 (Thr3/Ser10) Peptide TRF0211-M TRF0210-D TRF0210-M TRF0125-D TRF0125-M Note Go to 100 µg / 15,000 www.perkinelmer. com/kinaseselector 10 µg / 1,500 100 µg / 15,000 to view our kinase selector tool 0.5 nmole / 1,000 5 nmoles / 15,000 Additional items that need to be ordered with AlphaLISA and LANCE Ultra Methyl / Acetyl assays: Description Required for running AlphaScreen Streptavidin Donor beads AlphaLISA Assays AlphaLISA Biotin-anti-Histone H3 (C-ter) Antibody (required for full-length histone substrates) AlphaLISA 5X Epigenetics Buffer 1 Kit (required for histone H3-derived peptidic substrates) Required for running LANCE Ultra ULight – Streptavidin LANCE Ultra Assays LANCE Detection Buffer, 10 X Part Number 6760002S 6760002 6760002B AL118C AL118M AL118R AL008C AL008F TRF0102-D TRF0102-M TRF0102-R CR97-100 Quantity 1 mg 5 mg 50 mg 2 µg 40 µg 200 µg 10 mL 100 mL 1 nmole 10 nmoles 100 nmoles 250 mL *Source of enzyme and substrate provided in detailed protocols available online at: www.perkinelmer.com/epigenetics **PerkinElmer also offers a wide selection of assays for non-histone phosphorylation events. For more information, visit www.perkinelmer.com/kinase For more information, please visit www.perkinelmer.com/epigenetics PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2011 PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. SureFire is a registered trademark of TGR BioSciences, Pty. Ltd. AlphaLISA and AlphaScreen reagents are for research use only. All other trademarks are the property of their respective owners. 009997_01 Printed in USA December 2011 Assay Points 2,000 10,000 100,000 500 10,000 50,000 1,000 10,000 100,000