EpiTitan™ Histone Peptide Array Description: EpiCypher's EpiTitan™ Histone Peptide Array platform is designed for rapid and high-throughput screening of effector protein, antibody and enzyme interactions with a comprehensive library of combinatorially-modified and biotinylated histone peptides immobilized on a streptavidin-coated glass slide. The peptides encompass over 95 unique modifications on the four core histones (H2A, H2B, H3 and H4) and several histone variants. Every EpiTitan™ Histone Peptide Array contains more than 265 histone peptides spotted 12 times each for high quality detection and analysis of antibody or protein binding or enzyme activity. Extensive validation and purification of peptides is performed prior to histone peptide array printing to ensure the highest quality product for your studies. EpiTitan™ is a dramatic improvement over our previous EpiGold™ platform, which was the gold standard in histone peptide arrays.
EpiTitan™ improvements include:
Capability to perform multiple experiments per array with the included multi-well gasket
Over 20 additional peptides and more unique modifications
Improved positive controls for epitope tags and primary antibodies
Enhanced scanning and analysis tools
EpiTitan™ Histone Peptide Array Uses:
Examine the selectivity and specificity of histone modification antibodies
Analyze the specificity of histone binding proteins
Identify substrates for histone-modifying enzymes
EpiTitan™ Histone Peptide Array Advantages:
Significantly reduced false-negatives and false positives, reducing time and cost of experiments
Perform two separate experiments on a single array using the new multi-well gasket
HPLC quality control of all peptides allows full confidence in peptide consistancy from array analysis to follow-on experiments
Our industry leading reproducibility of results provides confidence in readouts; lab-to-lab reproducibility allows verification and adoption of results
High quality histone peptide arrays provide conclusions at research stage and reduces errors arising from other peptide array platforms
EpiTitan peptides are not limted to 20 residues, as all peptides are synthesized and validated prior to array spotting
Up to 6 unique modifications per peptide allows for greater understanding and utility of peptide protein interaction analysis, compared to competing array platforms that have max of only 4
EpiTitan™ Histone Peptide Array Storage and Stability: EpiCypher EpiTitan™ Histone Peptide Arrays should be stored in a dry environment at 4°C and must be protected from light and dust. Proper storage will maintain the integrity of the histone peptide array for at least 4 months from the date of shipping.
EpiTitan™ Histone Peptide Array Quality Control: EpiTitan™ Histone Peptide Arrays are created in batches of 50 to 100 arrays, and throughout the batch process, several sample arrays are analyzed for proper spotting using the patented fluorescent tracer co-spotted with each peptide. Several arrays per batch are also tested for performance using a histone binding "efftector" protein with a known binding pattern and analyzed for activity.
EpiCypher Histone Peptide Array References:
Rothbart SB et al (2015). An Interactive Database for the Assessment of Histone Antibody Specificity. Mol Cell 59: 502-511. LINK
Ali M et al (2015). Molecular insight into inhibition of the methylated histone-plant homeodomain complexes by calixarenes. J Biol Chem. LINK
Zhang ZM et al (2015). An Allosteric Interaction Links USP7 to Deubiquitination and Chromatin Targeting of UHRF1. Cell Rep. LINK
Tong Q et al (2015). An acetyl-methyl switch drives a conformational change in p53. Structure 23: 322-331. LINK
Tong Q et al (2015). Structural plasticity of methyllysine recognition by the tandem tudor domain of 53BP1. Tong Q et al. 2015. Structure 23: 312-321. LINK
Rothbart SB et al (2015). An Interactive Database for the Assessment of Histone Antibody Specificity. Mol Cell 59: 502-511. Link
Greer EL et al (2014). A histone methylation network regulates transgenerational epigenetic memory in C. elegans. Cell Rep 7: 113-126. Link
Kim HS et al (2014). Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function. Cell Rep 6: 892-905. Link
Klein BJ et al (2014). The histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers. Cell Rep 6: 325-335. Link
Kinkelin K et al (2013). Structures of RNA polymerase II complexes with Bye1, a chromatin-binding PHF3/DIDO homologue. PNAS USA 110: 15277-15282. Link
Ali M et al (2013). Molecular basis for chromatin binding and regulation of MLL5. PNAS USA 110: 11296-11301. Link
Rothbart SB et al (2013). Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation. Genes Dev 27: 1288-1298. Link
Gatchalian J et al (2013). Dido3 PHD modulates cell differentiation and division. Cell Rep 4: 148-158. Link
Cai L et al (2013). An H3K36 Methylation-Engaging Tudor Motif of Polycomb-like Proteins Mediates PRC2 Complex Targeting. Mol Cell 49: 571-582. Link
Rothbart SB et al (2012). Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation. Nat Struct Mol Biol 19: 1155-1160. Link
Rothbart SB, Krajewski K, Strahl BD, Fuchs, SM (2012). Peptide microarrays to interrogate the histone code. Methods Enzymol 512: 107-135. Link
Fuchs SM, Krajewski K, Baker RW, Miller VL, Strahl BD (2011). Influence of combinatorial histone modifications on antibody and effector protein recognition. Curr Biol 21: 53-58. Link
Additional Resources for EpiTitan™ Histone Peptide Array:
Supplemental files for EpiTitan™ Histone Peptide Array:
EpiTitan™ Histone Peptide Array Manual
EpiTitan™ Annotated Peptide List v4
EpiTitan™ Array Grid v4
EpiTitan™ Histone Peptide Array Workflow: For detection of the interaction of an effector protein with peptides on the array as shown above, you need a primary antibody to the protein (or to an affinity tag) and a fluorescently-labeled (or HRP-conjugated for ECL detection) secondary antibody to the primary. This is much like the detection procedure employed in immunofluorescence miscroscopy. For analysis of histone antibody specificity, you need a primary antibody to a histone modification and a labeled (fluorescent or HRP-conjugated) secondary antibody recognizing the primary antibody. (Click on image to enlarge).
ECL or Fluorescence - EpiTitan™ Histone Peptide Array Data: Histone peptide array detection of the binding specificity of Histone H3 K4Me3 Antibody (Catalog No. 13-0004). Left Panel: Flourescence scan of antibody binding. The patented spotting tracer appears green, and H3 K4Me3 binding is shown in red. Right Panel: ECL detection of K4me3 antibody binding visualized by 30-second exposure to X-ray film. (Click on image to enlarge).
EpiTitan™ Histone Peptide Array Data-Histone Methyltransferase Enzyme Assay: A recombinant histone lysine 9 methyltransferase was used to methylate peptides on the EpiTitan™Histone Peptide Array. Left Panel (-KMT): No enzyme control. Right panel (+KMT): Enzyme applied to array. Methylation was detected using an antibody recognizing H3K9me2. White boxes highlight H3K9 methylated peptides detected by the antibody prior to enzyme addition. New spots detected depict novel sites of H3K9me2 on the array. These results highlight the use of the EpiTitan™ Histone Peptide Array for use in detecting the substrate specificity of histone modifying enzymes. (Click on image to enlarge).
EpiTitan™ Histone Peptide Array Design: Each EpiCypher EpiTitan™ Histone Peptide Array contains over 265 biotinylated histone peptides (20 amino acids in length or more) immobilized on a streptavidin-coated glass slide. Peptides are spotted as two identical sub-arrays, labeled A and B. Within each sub-array, each peptide is spotted twice in groups of 3 (red dots), for a total of 12 spots per peptide on each array. (Click on image to enlarge).