1. Distributor Training Product Overview 2013

2. Agenda Provide background information on epigenetics as it relates to DNA methylation, histones, & chromatin Discuss ways to identify potential customers Talk about the current trends and techniques in each research area Go through the available Active Motif products and explain which product is best suited for the customer’s needs 2

3. Epigenetics Epigenetics is defined as the study of heritable and reversible changes in gene function that occur without a change in the DNA sequence What do Epigenetic modifications do? –Provide an additional layer of transcriptional control that regulates how genes are expressed –They are required for normal cell differentiation –Changes to epigenetic patterns are involved in cancer, autoimmune diseases and aging 3

4. Epigenetics Examples of Epigenetic Modifications: –Methylation (occurs on DNA and histones) –Acetylation –Phosphorylationoccurs on histones –Ubiquitination 4

5. DNA Methylation DNA Methylation occurs when a DNA methyltransferase enzyme (also called a DNMT) adds a methyl group (CH 3 ) to the carbon 5 position of a cytosine residue 5

6. Facts about DNA Methylation Definition: It is a chemical, heritable and reversible modification of the DNA –Added by DNA methyltransferases (DNMTs) –Removed by ? (TET enzymes is one theory) Location: DNA methylation is most commonly associated with a CpG sequence context. More recently non-CpG DNA methylation (CpA or CpT) has been found in embryonic stem cells Role: The presence of DNA methylation is associated with transcriptional repression 6

7. Role of DNA Methylation The lack of DNA methylation on a gene promoter enables gene expression to occur The presence of DNA methylation on a gene promoter prevents gene expression from occurring 7

8. Why is DNA Methylation important? It controls gene regulation –Suppression of repetitive elements –Tumor suppression Proper methylation is essential for normal development –Genomic imprinting –X-chromosome inactivation Aberrant DNA methylation is associated with developmental defects and cancer 8

9. Who is studying DNA Methylation? Researches focused on the following fields may be studying the effects of DNA methylation –Cancer –Aging –Neurobiology (development, psychiatry, addiction) –Stem cells and regenerative medicine –Hematopoiesis –Autoimmunity Labs focused in these research areas are potential customers 9

10. How can you find researchers? By utilizing a publication database and selecting keywords, it is possible to identify researchers publishing on DNA methylation An example of a publication database is Highwire –http://highwire.stanford.edu 10

11. How can you find researchers? Select “more search option” to expand the search criteria 11

12. How can you find researchers? Filter by keywords and/or publication dates –“DNA Methylation, Active Motif, Tokyo” –This will find researchers working on DNA methylation that have used an Active Motif product and are located in Tokyo 12

13. How can you find researchers? 13

14. How can you find researchers? To find new customers alter the search words –“DNA Methylation, Aging, Korea” –This will identify researchers working on DNA methylation in relation to aging located in Korea 14

15. How can you find researchers? 15

16. How can you find researchers? 16

17. How can you find researchers? Attending key meetings is another way to identify researchers working on Epigenetics Meetings focused on epigenetics, DNA methylation and chromatin will have the best research audience for Active Motif products 17

18. Current Trends & Techniques in DNA Methylation

19. How do you study DNA methylation? Traditional DNA methylation studies involve –Studying DNMT activity or inhibition –Comparing the methylation status of different samples (e.g. normal vs. diseased) –Understanding the methylation profile of a particular gene sequence Recently, new modifications of DNA methylation have been discovered –This requires the use of new techniques to distinguish between the different modifications 19

20. What is new in DNA Methylation? New DNA methylation modifications The role of these new modifications are currently of great interest to researchers NH 2 N N H O OH 5-hydroxymethylcytosine NH 2 N N H O 5-formylcytosine O H NH 2 N N H O 5-carboxylcytosine O OH 20

21. 5-Hydroxymethylcytosine (5-hmC) 5-hmC is derived from the oxidation of 5-mC by the TET family of cytosine oxygenases It is possible that 5-hmC may be an intermediary structure in DNA demethylation 5-hmC has been found in abundance in neuronal cells and embryonic stem cells 21

22. 5-Formylcytosine (5-fC) & 5-Carboxylcytosine (5-caC) 5-formylcytosine and 5-carboxylcytosine are the newest DNA modifications to be published Both 5-fC and 5-caC are derived from continued activity of the TET enzymes on 5-hmC Both 5-fC and 5-caC have been detected in the mouse male pronucleus following fertilization and are gradually diluted by DNA replication Inoue A et al. (2011) Cell Res. 21: 1670-1676. 22

23. DNA Methylation Products Available from Active Motif

24. How can Active Motif help in the study of DNA Methylation? Active Motif offers products to study –Traditional 5-Methylcytosine methylation –5-Hydroxymethylcytosine methylation –5-Formylcytosine & 5-Carboxylcytosine methylation –3-Methylcytosine methylation Active Motif’s products include –Assay kits –Antibodies –Epigenetic Services 24

25. How can Active Motif help in the study of DNA Methylation? 25

26. How can Active Motif help in the study of DNA Methylation? 26

27. How can Active Motif help in the study of DNA Methylation? Active Motif offers a variety of marketing literature pieces to describe our kits and reagents Literature can be downloaded from website as a pdf file www.activemotif.com/receiv e-product-information 27

28. Assays for DNA Methylation

29. Bisulfite Conversion/Sequencing Method –Unmodified cytosine residues are chemically converted to uracils, while methylated cytosines are protected from the chemical reaction and remain cytosines –Converted DNA is often analyzed by sequencing or with methyl specific PCR primers –Sequencing of bisulfite converted DNA enables the exact location of the methylated residue to be known 29

30. Bisulfite Conversion/Sequencing Customers –Studying single nucleotide resolution of DNA to identify the cytosine in the sequence that contains the methyl mark –Since whole-genome sequencing is very costly, customers may perform locus-specific analysis or comparison of different samples –Technique does not distinguish the type of methylation present (it cannot differentiate 5-mC from 5-hmC) Available Active Motif Products: Cat# 55001MethylDetector™50 rxns 30

31. Bisulfite Conversion/Sequencing Competition 31 CompanyProductFeatures Active MotifMethylDetector  Works with 200 pg – 2 µg DNA  99% conversion efficiency  5 hour conversion time  Includes control PCR primers Zymo ResearchEZ DNA Methylation Kit  Works with 500 pg – 2 µg DNA  99% conversion efficiency  Depending on the kit used, conversion time is 3-16 hr QiagenEpiTect Bisulfite  Works with as little as 1 ng DNA including FFPE DNA  99% conversion efficiency  6 hour conversion time

32. Methylated DNA enrichment Methylated DNA Enrichment –Is a technique used to select the subset of a genomic DNA population that contains methylation –Only DNA fragments containing the methylation will be present in the final elution –This reduces the complexity of the DNA population and thereby reduces the amount of money that needs to be spent on downstream applications such as sequencing Methods for Methylated DNA Enrichment: –Methylated DNA Immunoprecipitation (MeDIP) –Methyl-CpG-binding proteins (MBD) –Chemical labeling 32

33. Methylated DNA enrichment MeDIP: Methylated DNA Immunoprecipitation –Is an enrichment technique to isolate methylated DNA fragments from unmethylated DNA using an antibody specific for 5-methylcytosine in combination with protein G magnetic beads –hMeDIP is done using an antibody specific for 5-hydroxymethylcytosine 33

34. Methylated DNA enrichment Methylated DNA Immunoprecipitation (MeDIP) Customers –Want to reduce sample complexity to methylated DNA fragments –Can be studying either CpG or non-CpG methylation (works for stem cell researchers) –MeDIP only works with single-stranded DNA fragments while hMeDIP works with double-stranded DNA fragments –Technique specific for the antibody used (5-methylcytosine Ab) Available Active Motif Products: Cat# 55009MeDIP10 rxns Cat# 55010hMeDIP10 rxns 34

35. Methylated DNA enrichment Competition 35 CompanyProductFeatures Active MotifMeDIP hMeDIP  Use 100 ng – 1 µg ssDNA  Includes a bridging antibody to improve binding efficiency  Overnight capture  Includes control DNA and primers  Works on dsDNA Zymo ResearchMethylated DNA-IP  Use 50 ng – 500 ng ssDNA  3 hour capture  Includes control DNA and primers DiagenodeMeDIP hMeDIP  Requires 1 µg ssDNA  4 hour – overnight capture  Includes control DNA and primers

36. Methylated DNA enrichment Methyl-CpG Binding Proteins (MBD) –Isolate methylated DNA fragments from unmethylated DNA by using a protein specific for CpG methylation in combination with nickel magnetic beads 36

37. Methylated DNA enrichment Methyl-CpG Binding Proteins (MBD) Customers –Want to reduce sample complexity to methylated DNA fragments –This method requires the methylation to occur in a CpG context –Works with double-stranded DNA fragments –Technique specific for 5-mC (will not bind DNA with 5-hmC) Available Active Motif Products: Cat# 55005MethylCollector™ Ultra30 rxns 37

38. Methylated DNA enrichment Competition 38 CompanyProductFeatures Active MotifMethylCollector Ultra  Works with 1 ng – 1 µg dsDNA  Uses MBD2/MBD3 protein complex which has higher binding affinity than MBD2 protein alone  Includes control DNA and PCR primers  Elution as a single fraction to collect both low and high methylation targets InvitrogenMethylMiner  Uses MBD2 protein alone  Fractionated elution protocol to gradually elute methylated targets DiagenodeMethylCap  Uses MeCP2-GST fusion protein

39. Methylated DNA enrichment Chemical labeling (5-hmC specific technique) –5-hmC can be enriched through a chemical labeling process in which a modified glucose is attached to the hydroxymethyl modification –The modified glucose is conjugated to biotin –The biotin labeled 5-hmC residues can be selectively enriched with streptavidin magnetic beads –The system is sensitive to detect as little as two hydroxymethylcytosine residues –This technology was featured in a recent publication Song, C.X. et al. (2011) Nature Biotechnology 29: 68-72. 39

40. Methylated DNA enrichment Hydroxymethyl Collector™ 40

41. Methylated DNA enrichment Chemical labeling of 5-hmC Customers –Want a highly sensitive method to reduce sample complexity to contain only hydroxymethylated DNA fragments –Need to detect a small number of 5-hmC residues –Can be studying either CpG or non-CpG methylation –Works with double-stranded DNA fragments –Technique specific for 5-hmC Available Active Motif Products: Cat# 55013Hydroxymethyl Collector™25 rxns 41

42. Methylated DNA enrichment Competition –No competition –Only company to sell product using this methodology 42

43. Methylated DNA enrichment summary To help select the right enrichment kit 5-hmC 5-mC 5-C 5-hmC 5-mC Target 43

44. Associated DNA Methylation products DNMT Activity / Inhibition Assays is designed to study nuclear extract or purified proteins for DNA methyltransferase activity or to screen chemical compounds for the ability to inhibit DNMT activity Available Active Motif Products: Cat# 55006DNMT Activity / Inhibition Assay1 x 96 rxns 44

45. Associated DNA Methylation products Methylated DNA Standard Kit can be used as a control for DNA methylation experiments –Includes 3 DNA standards: unmethylated, methylated and hydroxymethylated DNA Methylation enzymes 45 Cat# 55008Methylated DNA Standard Kit3 x 2.5 µg Cat# 31363Recombinant Tet1 protein25 µg Cat# 55012  -Glucosyltransferase 500 units Cat# 55011PvuRts1I restriction enzyme50 units

46. Associated DNA Methylation products Antibodies (visit website for a complete list) –5-methylcytosine –5-hydroxymethylcytosine –5-formylcytosine –5-carboxylcytosine –3-methylcytosine –DNMT1 –DNMT 3A –DNMT 3B –MBD2 –MeCP2 46

47. Active Motif’s products for Chromatin

48. Chromatin Immunoprecipitation (ChIP) ChIP is a technique used to study protein-DNA interactions that occur within the cell It can be used to identify proteins that are actively bound to the DNA in its chromatin context When combined with sequencing, it can also be used to identify the DNA binding sequences of target proteins (ChIP-Seq) To preserve the relationship between the protein and the DNA, ChIP is performed using cells that have been fixed with formaldehyde 48

49. Chromatin Immunoprecipitation (ChIP) How does ChIP work? 49 qPCR Chromatin ChIP Purification Validation Sequencing Preparation microarray EpiShear™ Sonicator Enzymatic & Sonication Shearing Kits Ready-to-ChIP chromatin ChIP-IT™ Express ChIP-IT™ High Sensitivity ChIP-IT-Seq Re-ChIP-IT™ Chromatin IP DNA Purification Kit ChIP control Kits qPCR Analysis Kit qPCR primer sets

50. Chromatin Preparation ChIP starts with cells or tissues that are fixed with formaldehyde to cross-link the protein-DNA interactions and preserve the binding within the sample Cells are then lysed to release the fixed chromatin The chromatin is sheared into smaller fragments (200-1200 bp) using either sonication or enzymatic digestion 50

51. Chromatin Preparation EpiShear™ Sonicators Probe sonicator can be used to prepare chromatin from individual samples –Use 0.2 ml – 50 ml volume –Programmable settings Multi-Sample sonicator can process up to 8 vials simultaneously –Cup horn sonicator –Use 20 µl – 1.2 ml volume 51

52. Chromatin Preparation EpiShear Sonicators Customers –Labs who will perform chromatin immunoprecipitation using sonication as the shearing method –The probe sonicator can be combined with our cooled sonication platform, which allow the precise position of the sonicator probe and the sample temperature to be tightly controlled to ensure sample-to-sample reproducibility Available Active Motif Products: Cat# 53052EpiShear Probe Sonicator230V Cat# 53073EpiShear Cooled Sonication Platform1.5 ml Cat# 53063EpiShear Multi-Sample Sonicator with chiller230V 52

53. Chromatin Immunoprecipitation (ChIP) ChIP reactions utilize an antibody against the target protein of interest to immunoprecipitate the protein- DNA binding complex 53

54. Chromatin Immunoprecipitation (ChIP) Active Motif offers several kits to perform ChIP. To help identify the right kit for the experiment, consider the following conditions: Sample type – cultured cells or tissue samples Amount of sample – Cell equivalents to be used Abundance of target protein – high abundance protein like a histone or low abundance protein such as a transcription factor Speed of assay – 1 day or 3 day protocol Downstream application – qPCR or Sequencing 54

55. Chromatin Immunoprecipitation (ChIP) ChIP-IT Express –Ideal for use on cultured cells –Requires greater than 100,000 cell equivalents per IP –Good for ChIP of high abundance target proteins such as histones –Fast 1 day protocol uses magnetic Protein G beads for capture of protein-DNA complexes –Kit does not include DNA purification reagents –Enriched DNA can be used in downstream applications such as qPCR and sequencing 55

56. Chromatin Immunoprecipitation (ChIP) ChIP-IT Express Customers –Are working with robust antibodies to highly abundant protein targets –Sample material is not limiting such that greater than 100,000 cell equivalents can be used per IP –Time is a concern and they need results quickly –Have the option of performing chromatin shearing using either sonication or enzymatic digestion 56 Cat# 53008ChIP-IT Express25 rxns Cat# 53009ChIP-IT Express Enzymatic25 rxns

57. Chromatin Immunoprecipitation (ChIP) ChIP-IT High Sensitivity –Ideal for use on cultured cells and tissue samples –Works with as little as 1,000 cell equivalents for histones and 50,000 cell equivalents for transcription factor proteins –Good for ChIP of transcription factors or for use with ChIP antibodies with low binding affinity –3 day protocol uses Protein G agarose beads and a filtration column to avoid any loss of sample –Kit includes DNA purification reagents –Enriched DNA can be used in downstream applications such as qPCR and sequencing 57

58. Chromatin Immunoprecipitation (ChIP) ChIP-IT High Sensitivity 58

59. Chromatin Immunoprecipitation (ChIP) ChIP-IT High Sensitivity Customers –Are trying to ChIP low abundance transcription factor proteins and therefore need high sensitivity with very low background –May be working with ChIP antibodies with low binding affinity –Are working with limited cell numbers –Are working with tissue samples –Are willing to perform a longer assay (3 days) for improved sensitivity 59 Cat# 53040ChIP-IT High Sensitivity16 rxns

60. Chromatin Immunoprecipitation (ChIP) ChIP-IT-Seq (coming soon) –Ideal for use on cultured cells and tissue samples –Recommend to use 4.5 million cell equivalents for ChIP-Seq –Can be used with both high abundance and transcription factor target proteins –3 day protocol uses Protein G agarose beads and a filtration column to avoid any loss of sample –Kit includes enzymes and a protocol for library preparation of ChIP DNA for Next-Generation sequencing on the Illumina® GAII, HiSeq or MiSeq 60

61. Chromatin Immunoprecipitation (ChIP) ChIP-IT-Seq (coming soon) 61 Coming SoonChIP-IT-Seq10 libraries

62. Chromatin Immunoprecipitation (ChIP) Re-ChIP-IT –Is designed to perform sequential ChIP reactions using two different antibodies –It assays for the simultaneous presence of two proteins at the same genomic region of interest –Designed for use with high abundance protein targets –Works on greater than 750,000 cell equivalents –Does not include DNA purification reagents –Enriched DNA is good for use in qPCR 62 Cat# 53016Re-ChIP-IT25 rxns

63. ChIP DNA Purification Chromatin IP DNA purification Kit –Designed to purify ChIP DNA following enrichment –Purified DNA is required for use in downstream application such as qPCR and sequencing –Column based purification method gives equivalent yield results as phenol/chloroform extraction followed by ethanol precipitation –Includes pH indicator dye to ensure the binding reaction occurs at the correct pH 63 Cat# 58002Chromatin IP DNA Purification50 rxns

64. ChIP Accessory Products qPCR Analysis Kit –For use with the ChIP-IT High Sensitivity and ChIP-IT- Seq Kits for chromatin preparation –Provides recommendations to determine if the quality of the enriched chromatin is of sufficient quality for downstream sequencing or microarray applications –Includes Excel spreadsheet that automatically normalizes ChIP data to enable multiple samples and experiments to compared side-by-side 64 Cat# 53029ChIP-IT qPCR Analysis Kit10 rxns

65. ChIP Accessory Products qPCR Analysis Kit 65

66. ChIP Accessory Products qPCR primer sets –Available for human, mouse, rat and other species –Primers have been validated to work in qPCR –Both positive control primer and negative control primer sets are available to help confirm specificity of ChIP enrichment –www.activemotif.com/chipprimerswww.activemotif.com/chipprimers ChIP control kits –Available for human, mouse and rat –Include positive and negative control antibodies and PCR primer sets to validate new ChIP antibodies 66

67. ChIP Competition 67

68. Summary

69. Active Motif has a large selection of assay kits and antibodies available for the analysis of DNA methylation, including the most recently discovered modifications Active Motif offers assays kits for every step of the Chromatin immunoprecipitation workflow, from preparation of the chromatin through to validation of the enriched ChIP DNA 69

70. DNA Methylation Products Antibody immunoprecipitation of methylated DNA –MeDIP (5- mC) –hMeDIP (5-hmC) MBD enrichment of CpG DNA –MethylCollector ™ Ultra Unmethylated DNA enrichment –Unmethylcollector™ Chemical labeling and enrichment of 5-hmC –Hydroxymethyl Collector™ Methyl-sensitive enzymes –  -Glucosyltransferase –PvuRts1I –TET1 Bisulfite conversion –MethylDetector ™ DNA methyltransferase activity assay –DNMT Activity / Inhibition Assay Antibodies –5-methylcytosine –5-hydroxymethylcytosine –5-carboxylcytosine –5-formylcytosine –3-methylcytosine Methylated DNA controls –Methylated DNA Standards –Fully methylated Jurkat DNA 70

71. ChIP Products Chromatin Preparation –EpiShear Probe Sonicator –EpiShear Cooled Sonication Platform –EpiShear Multi-Sample Sonicator –Ready-to-ChIP chromatin ChIP Kits –ChIP-IT Express –ChIP-IT Express Enzymatic –ChIP-IT High Sensitivity –ChIP-IT-Seq –Re-ChIP-IT –ChIP-IT Express High Throughput –RNA-ChIP-IT ChIP DNA Purification –Chromatin IP DNA Purification Kit ChIP Accessory Products –ChIP-IT qPCR Analysis Kit –ChIP-IT Control Kits –ChIP control qPCR primer sets –Protein G magnetic beads –Protein G agarose beads –Bridging antibody for Mouse IgG Services –Epigenetic Services 71

72. Thank You!