1. Histone Analysis Product Overview 2012

2. Histones Histones are alkaline proteins (H2A, H2B, H3 and H4) that package DNA into structural units called nucleosomes. Primary protein components of chromatin. 147 bp DNA winds around octameric nucleosome core particle consisting of dimers of each core histone. Play a significant role in gene regulation Undergo post-translational modifications (acetylation, methylation, phosphorylation, etc) 2

3. Why are Histones important? It controls gene regulation (‘Histone Code’) –Regulates binding/accessibility of regulatory proteins and transcriptional machinery via: »Chromatin compaction »Histone modifications (ie active-H3K4me3, repressed- H3K27me3, poised/bivalent-both) Aberrant histone regulation is associated with improper gene expression and DNA damage and can lead to developmental defects and cancer 3

4. Histone Analysis Histone Purification Histone Peptide Array Histone Modification ELISAs Recombinant Histones HAT HDAC HDM Histone modifying enzymes Histone antibodies 4

5. Histone Purification Kits

6. Histone Purification Kit Customer –Interested in obtaining pure fractions of histone proteins while preserving post-translational modifications –Histones are not isolated under normal nuclear extraction procedures. They require acid extraction Format –Two kit types available: Histone Purification Kit contains 10 rxns Histone Purification Mini Kit contains 20 rxns 6

7. Histone Purification Kits Histone Purification Kit –Single fraction of H2A, H2B, H3 & H4 proteins –Or fractions of H2A/H2B and H3/H4 proteins Histone Purification Mini Kit –Single fraction of H2A, H2B, H3 & H4 proteins –Works from 8x10 6 cells or 1g of tissue –Fast and easy 7

8. Histone Purification Kits Advantages –Preserves post-translational modifications –Fast –Works on cell and tissue samples –Allows elution of histones together in one fraction or as separate H2A/H2B and H3/H4 fractions –Histone purification kit provides better input sample for Histone Modification ELISAs than acid extraction protocol Competitors : –Epigentek EpiQuik (or Abcam EpiSeeker) Total Histone Extraction Kit – 100 rxn format, only extracts total histone fraction. –This method is patented, so we have an advantage with our exclusive kit! 8

9. MODified™ Histone Peptide Array

10. 384 Different histone modification combinations 59 post-translational modifications for acetylation, phosphorylation, methylation and citrullination Peptides are 19mer long with up to four separate modifications on each peptide Excel sheet on website as reference for spots and their location on array 10

11. MODified™ Histone Peptide Array Customers –Characterizing antibody cross-reactivity –Screening for protein or enzyme interactions with histone modifications Format –Single array –5 pack array MODified Array Labeling Kit available –Contains buffers, c-Myc control antibody, rabbit and mouse HRP-conjugated secondary antibodies and ECL reagents for chemiluminescent detection of MODified Peptide Array 11

12. MODified™ Histone Peptide Array 12

13. MODified™ Histone Peptide Array Free software available online –Array Analyse Software can be downloaded from website –Software only works on PCs 13

14. MODified™ Histone Peptide Array Advantages –Histone specific panel testing H2A, H2B, H3 & H4 –Up to 4 modifications per 19mer peptide enables analysis of the effects of neighboring modifications on site recognition and binding –Detects like a Western blot (Colorimetric or Chemiluminescent) –Works with antibodies, proteins or enzymes 14

15. MODified™ Histone Peptide Array Lake Placid has tested some of our top selling antibodies - this data will be added to the CofA and website as available To see this data go to www.activemotif.com/arraytested Array has been validated for use with histone modifying enzymes, such as HMT (data available in product manual) Data for 39161 Histone H3 trimethyl Lys9 pAb is shown below 15

16. Histone ELISA kits

17. Histone Modification ELISAs Visit www.activemotif.com/hiselisa for a current listwww.activemotif.com/hiselisa Current available Histone ELISA kits : Total Histone H3 ELISA Histone H3 mono-, di- and tri-methylated Lys4 (H3K4) ELISAs Histone H3 di- and tri-methylated Lys9 (H3K9) ELISAs Histone H3 mono- and tri-methylated Lys27 (H3K27) ELISAs Histone H3 acetyl Lys9 (H3K9) ELISA Histone H3 acetyl Lys14 (H3K14) ELISA Histone H3 phospho Ser10 (H3 pS10) ELISA Histone H3 phospho Ser28 (H3 pS28) ELISA 17

18. Histone Modification ELISA Flow Chart 18

19. Who are our customers? -Researchers involved in epigenetics and histone modifications -customers wanting to quantitate methylation of lysine residues within their samples. -Customers currently using our histone purification kits (Catalog Nos. 40025 & 40026) as well as our modification specific histone antibodies may be interested in this product. Questions to ask : - Are you currently studying histone modifications such as lysine methylation? - Are you currently performing immunoblotting experiments to do this analysis? - Are you trying to quantitate the amount of histone methylation in your samples? - Are you trying to normalize for total histone H3 levels? 19

20. Histone ELISA kits : benefits Modification specific antibodies that do not cross-react with other site- or degree-specific methylation Recombinant protein control (positive control included) for quantification with site- and degree-specific lysine methylation Optimized buffers and protocol Lower price than other kits on the market Multiple modifications to chose from Can compare dimethyl Lys9 with trimethyl Lys9, or normalize against Total Histone H3. Additional modifications anticipated for release this year. Less than 4 hours 20

21. Histone ELISA kits : competitors CompanyProductWeakness Cell SignalingPathScan dimethyl Lys36 PathScan dimethyl Lys4 PathScan dimethyl Lys9 PathScan monomethyl Lys4 PathScan pan-methyl Lys9 PathScan trimethyl Lys27 PathScan trimethyl Lys4 PathScan Total Histone H3 PathScan phospho Histone H3 Ser 10 Also has acetyl modification kits No positive control, assay takes 4 hrs to overnight to complete Invitrogen (Biosource assay) Histone H3 (Total) Histone H3 phospho Ser10 Limited product offering Bethyl LabsHistone H3 PathProfiler Kit (either Total H3 or Phospho Ser10) Limited product offering, no positive control 21

22. Recombinant Histone Proteins

23. Available: –Recombinant unmodified histones –Recombinant methylated histones –Recombinant acetylated histones –Recombinant phosphorylated histones –Biotinylated histones for use in FRET assays Applications: –Can use these as substrates in functional assays, or in chromatin assembly kit to generate their own chromatin –Good substrates for Pharma experiments 23

24. Generation of Recombinant Histones Prepared using one of two patented technologies: –Expressed Protein Ligation (EPL) »A semisynthetic method in which an expressed protein containing C- terminal thioester is ligated to a peptide containing an N-terminal cysteine via a chemoselective reaction. »Preserves the native amine bond and natural protein structure, more closely mimics natural histones. –Methylated Lysine Analog (MLA) »A chemical alkylation reaction that substitutes aminoethylcysteine, a lysine analog that is structurally and chemically similar to lysine, for the existing lysine at the desired residue. »Provides precise control over the site and degree of methylation. Despite the substitution, recombinant proteins show functional similarity to native proteins. 24

25. Recombinant Histone Proteins Dot blot was used to confirm the identity of H3K79me2 recombinant protein 1 µg of various recombinant proteins were spotted onto a PVDF membrane Histone H3 dimethyl Lys79 pAb (Cat # 39143) was used at a 1:1,000 dilution 25

26. HAT (Histone Acetyltransferase) Assay

27. What is HAT? Histone AcetyTransferases are enzymes that put acetyl- modifications on histone tails. Ex: p300 and PCAF Role in gene regulation. Involved in cancers and disease and are the targets of new drug development. 27

28. HAT Assay Kit Customers –Academic researcher studying Chromatin biology –Pharma companies studying histone acetyltransferase inhibitors or screening inhibitors as potential drug compounds Format –96 reactions –Fluorescent assay (ex. 360-390nm, em. 460-490nm) –Includes enough p300 catalytic domain protein to screen 96 wells of inhibitor compounds –Includes anacardic acid as a control inhibitor –H3 and H4 N-terminal peptides are included as HAT substrates 28

29. Protocol Used for testing activity of a purified protein and inhibitor compounds. In a 96 well plate, add protein, (inhibitors),substrate, Acetyl-CoA, buffers, incubate 10 to 30 minutes at room temp, develop and read on plate reader. 29

30. HAT Assay Kit –Quick and sensitive method to screen for HAT activity or inhibition –Quantitative method when using standard curve –Can assay both H3 and H4 acetyltransferase activity –Includes control p300 protein and an inhibitor –This kit has not been optimized for cell lysates, however, it is possible (we did not test but it’s published) if the extracts are treated to remove free sulfhydryl groups in the sample. 30

31. Who would buy the HAT assay kit? Someone who is trying to target HAT activity in a disease pathway and needs to test treatments. Someone who has isolated a new protein that may be another Histone Acetyltransferase and needs to test activity. Someone who is working with HDACs may also need to test for HAT activity. Why? - To screen inhibitors of HAT activity - To test activity of a new purified HAT 31

32. Competitors and Sales Strategy Primary competitors: –Upstate (Millipore), but this is a sandwich ELISA format –Formats like ours: BioVision, Cayman Chemical, MBL international. Advantages: –Pricing, ours is cheaper. Our kit is very similar to the Cayman kit. They advertises theirs as an “inhibitor screening” assay; but, we tested it and it does not work to test inhibitor activity. Sales strategy: –Kit is similar to other kits already on the market such as BioMol Fluor- de-Lys assay –Cross sell with recombinant proteins: »Recombinant p300, catalytic domain »Recombinant GCN5 32

33. HDAC Assay Kit

34. What are HDACs? Histone Deacetylase enzymes remove acetyl groups from histone tails. Who studies HDACs? Academics, cancer researchers. HDACs are the target of many new screenings for drug compounds. 34

35. What is this kit? Colorimetric and fluorescent HDAC assays available. Used to test HDAC activity in nuclear extracts. 96-well plate format, HeLa nuclear extract included as a control. Kit contains all necessary buffers and substrates: –HeLa nuclear extract –Trichostatin A (HDAC activity inhibitor) –Acetyl-lysine substrate (colorimetric or fluorometric) –96-well assay plate 35

36. HDAC Assay Method + developing solution Reacts with the deacetylated substrate Substrate : acetylated peptide + HDAC sample + inhibitors Incubate at 37˚C for 30-60 minutes +Stop solution chromophore (color kit) or fluorophore (fluor kit) Measure the signal intensity 36

37. HDAC Assay Kits Advantages –Works with either nuclear extracted samples, purified recombinant HDAC enzymes or enzymes isolated by immunoprecipitation –Available in either fluorescent or colorimetric formats –Quantitative method when using the standard curve –Includes a control extract and inhibitor Can bundle with HAT assay, nuclear extract kit, HDAC antibody customers Customers –Chromatin biology –Pharma companies studying HDAC inhibitors 37

38. Histone Demethylase Assay Kit

39. What are HDMs? Histone Demethylase enzymes remove methyl groups from histone tails. LSD1 and Jmj; LSD1 specifically demethylates H3K4, an important mark associated with transcriptional activation. Who studies HDMs? Academics, cancer researchers. LSD1 is important target for drug discovery. The Histone Demethylase Assay is used to screen activity of purified or recombinant lysine-specific demethylase (LSD1) enzymes. Customers –Academic researcher studying Chromatin biology –Pharma companies studying histone demethylase inhibitors or screening inhibitors as potential drug compounds 39

40. What is this kit? Fluorometric 96-well plate format. Assays activity/inhibition of purified or recombinant LSD1 enzyme. Can be used to analyze overall histone conversion efficiency of dimethyl substrate to either mono- or un-methylated state. Kit contains all necessary buffers and substrates: –Control LSD1 enzyme –Recombinant Histone H3K4me2 substrate –Demethylation standard for quantification –96-well black half area assay plate How does it work? –LSD1 demethylation of the H3K4me2 substrate causes release of formaldehyde at each step, which reacts with the Detection reagent and generates a fluorescent signal 40

41. HDM Assay Protocol 41

42. HDM Assay kit : competitors CompanyProductWeakness Arbor AssaysDetectX Histone Demethylase Fluorescent Activity Kit Includes both Jmj-type and LSD1 HDMs. However, uses H3K4me2 peptide substrate BPS Bioscience LSD1 Fluorescent Assay KitMeasures hydrogen peroxide Uses a methylated peptide substrate Works with LSD1 only Biomol - Enzo Life Sciences LSD1 Fluorimetric Drug Discovery KitMeasures hydrogen peroxide Uses a H3K4me2 peptide substrate Works with LSD1 only Ex. 530, Em. 590 Epigentek (Kit also sold by Abnova) EpiQuick Histone Demethylase LSD1 Activity/Inhibition Assay Antibody-based assay that provides a fluorescent readout that is inversely proportional to HDM activity (negative assay). Both capture and detecting antibodies are used. Fluorescent settings indicate a hydrogen peroxide detection Uses a H3K4me2 peptide substrate Works with LSD1 only Ex. 530, Em. 590 42

43. HDM Assay Kits Advantages –The formaldehyde excitation/emission spectra allows for use of common filter sets (unlike hydrogen peroxide byproducts Ex 550/Em 590) –Only company to use a recombinant H3K4me2 substrate, mimics in vivo conditions better than peptide substrates. –Includes positive control LSD1 and standard for quantification –Fast, 2.5 hour protocol Cross-sell products include: –LSD1 enzyme, recomb. Methylated Histones, Histone ELISAs, Histone Purification Kits, Methylated Histone antibodies Customers –Chromatin biology, cancer, transcription biology, DNA repair –Pharma companies studying HDAC inhibitors 43

44. Histone Modifying Enzymes

45. Active Motif offers a variety of enzymes specific for modifying histones –Acetyltransferases –Deacetylases –Methyltransferases –Demethylases –Kinases For a complete list visit –www.activemotif.com/hismodenz 45

46. Histone Modifying Enzymes MODified Histone Peptide Array tested with histone methyltransferase G9a Top: 25 µM G9a (Cat. # 31327) and 1 mM AdoMet Bottom: 25 µM G9a mutant (Cat. # 31328) and 1 mM AdoMet The wild-type G9a protein was able to establish new methylation sites on the MODified Histone Peptide Array, as detected with the Histone H3 dimethyl Lys9 antibody 46

47. Chromatin assembly kit

48. Chromatin assembly kit : overview of the kit What is it? –This kit enables customers to assemble chromatin on linear or circular input DNA in vitro to be used for downstream assays (ie in vitro transcription and ChIP, histone mod assays.) –This kit is like a sample-preparation kit, this kit is not the experimental end-point. Why use the kit? –To investigate the effect of the chromatin configuration on nuclear function. Naked DNA just doesn’t always reveal the underlying mechanisms involved in gene regulation since it does not resemble the in vivo chromatin-like environment. How does it work? –The kit contains recombinant histone chaperone and chromatin assembly factor proteins, core histones, and ATP to assemble nucleosomes in an ordered array on the DNA sequence of interest. 48

49. Chromatin assembly kit : principle 49

50. Chromatin assembly kit : Applications In vitro transcription assays Shows what factors are required for transcription. In vitro histone acetyltransferase assays Shows if associated factors are required for histone acetylation. In vitro ChIP assays Shows what additional proteins associate with targeted factors and sequences. Could just be a control to show the in vitro chromatin resembles in vivo results. 50

51. Benefits/Competitors Results in an extended ordered array of nucleosomes. Saves researchers the time and headache of expressing and purifying the proteins for the assembly process themselves. Works with either linear or supercoiled DNA. Easy protocol: just mix together and incubate. Siliconized tubes and reagents to confirm the reaction worked are provided. Competitor: Vaxron, Millipore –Their kit is more expensive. –This kit does not result in an ordered array of nucleosomes. –Theirs also works with either linear or circular DNA (or PCR products). 51

52. Who are our customers? Researchers purifying histones Researchers working on chromatin structure  cancer research  transcription and influence of the chromatin on transcription  all researchers interested in epigenetics (cancer, developmental diseases, transcription, chromatin biology,retrovirology,…) 52

53. Antibodies

54. Histone Antibodies 170 antibodies against histones and specific histone modifications 155 antibodies against chromatin modifiers (HATs, HDACs, HDMs, etc) More added every month 54