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 caused by mechanisms that do not alter DNA sequence, 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 validate 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:

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

IN VITRO TOOL BOX

LANCE Ultra – robust, ultra high-throughput TR-FRET technology

AlphaLISA – high sensitivity, high throughput and ultimate flexibility - assay short peptides to full-length proteins

CELLULAR DETECTION KITS

AlphaLISA & Cell-Histone Buffers - rapid and direct detection of endogenous histone modulation in a one-well, no-wash assay format

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 simple 3-step protocol: 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

Cellular Detection Ordering and Assay Information

Mark Detected Part Number Assay Points

Methyl Modifications

H3K4me2 AL716HV 100 AL716C 500 AL716F 5,000

H3K27me3 AL722HV 100 AL722C 500 AL722F 5,000

H3K9me2 AL717HV 100 AL717C 500 AL717F 5,000

H3K27me2-1 AL721HV 100 AL721C 500 AL721F 5,000

H3K36me2 AL723HV 100 AL723C 500 AL723F 5,000

H3K79me2 AL748HV 100 AL748C 500 AL748F 5,000

Phospho Modifications

H3Ser10p* TGRH3S500 500 TGRH3S10K 10,000 TGRH3S50K 50,000

Normalization Assay

H3K4 unmodified AL719HV 100 AL719C 500 AL719F 5,000

Additional Items

AlphaScreen® General IgG (Protein A) 6760617C 500 Detection Kit 6760617M 10,000 6760617R 50,000

* Requires AlphaScreen General IgG (Protein A) Detection Kit

Read Plate on EnVision® or EnSpire®

Seed cells in plate4 hours

Treat cells – overnight

PREPARATIONOvernight

Add Cell-Histone Lysis Buffer15 min

Add Acceptor Beads and Biotinylated Anti-H360 min

Add Donor Beads 30 min

Add Cell-Histone Extraction Buffer10 min

EXTRACTION25 min

DETECTION90 min

ALL-IN-ONE WELL PROTOCOL

Mark Detected Part Number Assay Points

Acetyl Modifications

H3K9ac AL714HV 100 AL714C 500 AL714F 5,000

H3K27ac AL720HV 100 AL720C 500 AL720F 5,000

In Vitro Ordering and Assay Information

In Vitro Assay Toolbox

The in vitro toolbox comprises validated off-the-shelf reagents for the detection of post-translational modifications of histone H3, H4, and p53.

AlphaLISA and LANCE Ultra reagents to measure histone and other post-translational modifications

All detection reagents have been well characterized for their specificity at detecting the modification of interest, and validated for use with the specific enzymes depicted in the figure above.

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.

Aurora B

Cellular assayavailable in Alpha

G9a

P300

NH2 – AR2T3K4QTARK9S10TGGKAPRKQLATKAARK 27SAPATGGVK36KPHR. . .

SET7/9

Full-lengthprotein assay

available in Alpha

HDAC6HDAC3/NCOR2

HDAC2HDAC1SIRT3

HDAC3/NCOR1HDAC10

SIRT2SIRT1

JMJD2AHDAC1

EZH2

LSD1

HASPIN

methylation

demethylation

acetylation

deacetylation

phosporylation

CBP/p300 SIRT1N – p53 – K382Ac

PRMT6PRMT1

Writers

Erasers JMJD3

P300

JMJD1A

HDAC6HDAC3/NCOR2

HDAC2HDAC1

SIRT3SIRT7

HDAC3/NCOR1

HDAC10

SIRT2SIRT1

K

Excitation680 nm

Emission615 nm

B

Me

+ Enzyme

+ Eu-Ab+ ULight-SA

ULightEm 665 nm

SAM

SAH

K

K

K

Excitation320 or 340 nm

FRET

B

Eu

B

B

Me

Me

LANCE Ultra TR-FRET AlphaLISA Beads

Validated Enzyme Assays for Histone H3

Acetyl Modifications Validated Assays Alpha Technology LANCE Ultra (TR-FRET) AlphaLISA anti-mark Acceptor Beads LANCE Ultra Europium-labeled anti-mark Antibody Mark Detected Acetyltransferase Deacetylase (requires Alpha Streptavidin Donor beads) (requires LANCE Ultra ULight- Streptavidin) Part Number Quantity Assay Points Part Number Quantity Assay Points H3K4 SIRT1 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 H3K9/K27 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 H3K9ac p300 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 H3K27ac p300 HDAC1 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 H4Kac (pan) TIP60 AL143C 250 µg 500 TRF0412-D 10 µg 1,500 AL143M 5 mg 10,000 TRF0412-M 100 µg 15,625 AL143R 25 mg 50,000

Non-Histone Target p53 K382ac p300 SIRT1 AL124C 250 µg 500 TRF0409-D 10 µg 1,500 AL124M 5 mg 10,000 TRF0409-M 100 µg 15,625 AL124R 25 mg 50,000

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 EZH2 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 Histone H3 DOT1L AL147C 250 µg 500 C-terminus* AL147M 5 mg 10,000 AL147R 25 mg 50,000 H3R2me PRMT6 AL139C 250 µg 500 AL139M 5 mg 10,000 AL139R 25 mg 50,000 H3Rme and PRMT4 AL151C 250 µg 500 TRF0415-D 10 µg 1,500 H4Rme (pan) AL151M 5 mg 10,000 TRF0415-M 100 µg 15,625 AL151R 25 mg 50,000 H4K20me1 SET8 AL145C 250 µg 500 AL145M 5 mg 10,000 AL145R 25 mg 50,000 H4R3me PRMT1 AL150C 250 µg 500 TRF0414-D 10 µg 1,500 AL150M 5 mg 10,000 TRF0414-M 100 µg 15,625 AL150R 25 mg 50,000* The DOT1L assay requires also the biotinylated anti-H3K79me2 antibody (AL148)

Other Postranslational Modifications Validated Assays Alpha Technology LANCE Ultra (TR-FRET) AlphaLISA anti-mark Acceptor Beads LANCE Ultra Europium-labeled anti-mark Antibody Mark Detected (requires Alpha Streptavidin Donor beads) (requires LANCE Ultra ULight™-Streptavidin) Part Number Quantity Assay Points Part Number Quantity Assay Points H3Thr3p TRF0211-D** 10 µg 1,500 TRF0211-M 100 µg 15,625 H3Ser10p TRF0210-D** 10 µg 1,500 TRF0210-M 100 µg 15,625 O-GlcNAc O-GlcNAcase/OGA AL144C 250 µg 500 TRF0413-D 10 µg 1,500 AL144M 5 mg 10,000 TRF0413-M 100 µg 15,625 AL144R 25 mg 50,000 *Source of enzyme and substrate provided in detailed protocols available online at: www.perkinelmer.com/epigenetics**For LANCE Ultra kinase products, directly labeled substrates are available. PerkinElmer also offers a wide selection of biochemical and cellular assays for non-histone phosphorylation events. Please visit www.perkinelmer.com/kinase for more information.

Real-time Measurement of Enzyme Kinetics

Utilizing PerkinElmer's widely adopted mobility shift microfluidics assay technology, the LabChip® EZ Reader is ideal for profiling and mechanism of action studies for epigenetic research. This technology combines the advantages of capillary electrophoresis and microfluidics to offer direct measurement of substrate and product, rapid assay development and real time kinetic reads.

ProfilerPro fluorescent substrates are specifically designed for EZ Reader technology. Choose from multiple classes of enzyme targets.

Name Product Number Sequence Derived from

HDAC Substrates (1.5 mM, 0.5 mL in DMSO)

Histone 3 Acetylated Peptide 760511 FITC-TGGK(Ac)APR-COOH Histone H3 (aa. Pos 1-7)

p53 Acetylated Peptide 760512 FITC-TSPQPKK(Ac)-CONH2 p53 (aa. Pos 313-320)

Histone 4 Acetylated Peptide 760513 FITC-LGKGGAK(Ac)-CONH2 Histone H4 (aa. Pos 11-17)

LabChip Microfluidic Chips

HLabChip EZ Reader 12-Sipper Chip 760404

LabChip EZ Reader 4-Sipper Chip 760394

LabChip EZ Reader 152919

Additional items that need to be ordered with AlphaLISA and LANCE Ultra Methyl / Acetyl assays: Description Part Number Quantity Assay Points Required for running AlphaScreen Streptavidin Donor beads 6760002S 1 mg 2,000 AlphaLISA Assays 6760002 5 mg 10,000 6760002B 50 mg 100,000 AlphaLISA Biotin-anti-Histone H3 (C-ter) Antibody AL118C 2 µg 500 (required for full-length histone substrates) AL118M 40 µg 10,000 AL118R 200 µg 50,000 AlphaLISA Biotin-anti-H3K79me2 Antibody AL148C 2 µg 500 (required for the DOT1L assay with nucleosome substrates) AL148M 40 µg 10,000 AL148R 200 µg 50,000 AlphaLISA Biotin-anti-Histone H4 Antibody (required for full length histone substrates) AL146C 2 µg 500 AL146M 40 µg 10,000 AL146R 200 µg 50,000 AlphaLISA 5X Epigenetics Buffer 1 Kit AL008C 10 mL (required for histone H3-derived peptidic substrates) AL008F 100 mL Required for running LANCE Ultra ULight – Streptavidin TRF0102-D 1 nmole 1,000 LANCE Ultra Assays TRF0102-M 10 nmoles 10,000 TRF0102-R 100 nmoles 100,000 LANCE Detection Buffer, 10 X CR97-100 250 mL

For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs

Copyright ©2012-2013 PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. AlphaLISA and AlphaScreen reagents are for research use only. All other trademarks are the property of their respective owners. 009997B_01 October 2013

PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602www.perkinelmer.com

For more information, please visit www.perkinelmer.com/epigenetics

Ordering InformationTo choose the right product for your methylation assays, PerkinElmer offers several Adenosyl-L-Methionine, S-[methyl-3H] products varying in solvent, specific activity, concentration and package size.

Product SpecificActivity Concentration Size PartNumber

Adenosyl-L-Methionine, S-[methyl-3H] 5-15Ci(185-555GBq)/mmol 0.55mCi(20.35MBq)/mL 50uCi(1.85MBq) NET155050UC

5-15Ci(185-555GBq)/mmol 0.55mCi(20.35MBq)/mL 250uCi(9.25MBq) NET155250UC

5-15Ci(185-555GBq)/mmol 0.55mCi(20.35MBq)/mL 1.0mCi(37MBq) NET155001MC

Packaged in 10 mM sulfuric acid: ethanol (9:1). Shipped in dry ice.

Adenosyl-L-Methionine, S-[methyl-3H] 55-85Ci(2.03-3.15TBq)/mmol 0.55mCi(20.35MBq)/mL 250uCi(9.25MBq) NET155H250UC

55-85Ci(2.03-3.15TBq)/mmol 0.55mCi(20.35MBq)/mL 1.0mCi(37MBq) NET155H001MC

Packaged in 10 mM sulfuric acid: ethanol (9:1) under argon. Shipped in dry ice.

Adenosyl-L-Methionine, S-[methyl-3H] 12-18Ci(444-666GBq)/mmol 1.0mCi(37MBq)/mL 250uCi(9.25MBq) NET155V250UC

12-18Ci(444-666GBq)/mmol 1.0mCi(37MBq)/mL 1.0mCi(37MBq) NET155V001MC

Packaged in hydrochloric acid, pH 2.0-2.5, under argon. Shipped in dry ice.

3H-SAM Methylation Assay

Enhance your screening ability and advance your research by using the gold standard reagent for the study of histone methylation – Adenosyl-L-Methionine, S-[methyl-3H]; (3H-SAM). This reagent enables both quantitative kinetic measure of substrate methylation rates and the measure of methyltransferases with low activity. By using 3H-SAM, the labeled methyl group is directly incorporated without disrupting the biology of the reaction you’re studying. PerkinElmer NEN Radiochemicals, a proven leader since 1956.

Gold standard detection

• No antibodies • High sensitivity to detect low levels of enzyme activity • Direct methylation measurement • Reliable assay method for over 50 years