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Active Motif's Histone Analysis Brochure Histone Analysis Histone Analysis innovative tools designed to help unravel the histone code Histone Antibodies Histone Purification Active Motif’s unique portfolio of histone technologies provides researchers with a complete solution for the analysis of histones Recombinant Histones and their post-translational modifications, beginning with histone Histone Modifying Enzymes purification and continuing through to chromatin assembly. Histone Modification ELISAs Let Active Motif’s antibodies, enzymes and modification-specific Histone Demethylase Assay assays simplify your histone analysis. Histone Peptide Arrays Histone Binding Assay HAT/HDAC Assays Chromatin Assembly Histone Analysis – tools designed to help unravel the histone code Histone modifications such as acetylation, phosphorylation and methylation at specific amino acid residues on the histone globular domain and the N-terminal tails have been shown to influence and regulate transcription, chromosome packaging and DNA damage repair. Due to the importance of histone modifications in regulating chromatin structure and disease, Active Motif has developed a variety of products that simplify histone analysis. Histone Purification Active Motif’s Histone Purification and MW Log. Mitotic Sodium MW MW Core Histones Histone Purification Mini Kits enable you to Arrested Butyrate 98 98 isolate core histones from any cell culture 64 64 98 or tissue sample while preserving their post- 50 50 64 translational modifications. The kits use a 36 50 unique purification resin and a series of 36 36 proprietary elution buffers to isolate very 30 30 pure fractions of histones (Figure 1). 30 H3 16 H2B 16 H3 Unlike standard acid extraction techniques, it H2A H3 H4 H2B 16 is possible to isolate core histones as either H4 H2A/H2B H2A H4 a single fraction, or to further separate them 6 6 into H2A/H2B and H3/H4 fractions (Table 1). 6 Additionally, our unique extraction buffer 4 4 prevents unwanted enzymatic reactions from Figure 1: Core histone purified from HeLa cells and brain tissue. occurring, thereby preserving the existing Ten µg of sample were loaded per lane. Left: Core histones purified from logarithmically growing mitotic arrested histone modifications. or sodium butyrate-treated HeLa cells. Center: Separate H2A/H2B and H3/H4 fractions purified from HeLa cells. Right: Core histones isolated from rat brain tissue. Purified histones are ready for downstream analysis with Active Motif’s many different histone modification antibodies, or they can Kit Format Elution Capacity be used as substrates in functional assays, Histone Purification Kit Gravity Flow Separate H2A/H2B & 05-2.5 mg such as Active Motif’s Histone Modification H3/H4 fractions ELISAs and Chromatin Assembly Kits. Spin Column H2A, H2B, H3 & H4 in 0.5-2.5 mg a single fraction Histone Purification Mini Kit Mini Spin Column H2A, H2B, H3 & H4 in 0.1-0.5 mg a single fraction Advantages Table 1: Comparison of the original Histone Purification Kit and the Histone Purification Mini Kit. • Preserves modifications on histones, such as acetylation, phosphorylation and methylation To learn more about either the Histone Purification Kit or the Histone Purification Mini Kit, please call or visit us at www.activemotif.com/histonepur. • Purifies total core histones (H2A, H2B, H3 & H4) or separate fractions of H2A/H2B dimers and H3/H4 tetramers Product Format Catalog No. • Histone Purification Mini Kit can purify Histone Purification Kit 10 rxns 40025 histones from budding yeast Histone Purification Mini Kit 20 rxns 40026 On the cover: Ribbon diagram of the nucleosome core particle structure (H2A.Z nucleosome, pdb entry 1F66) viewed down the superhelical axis (left) and rotated 90° (right). Original figure was prepared by Dr. Karolin Luger, Department of Biochemistry and Molecular Biology, Colorado State University. www.activemotif.com MODified™ Histone Peptide Array & Analysis Software The MODified™ Histone Peptide Array* is a +' >_ijed[>)jh_c[j^obBoi/f7X valuable research tool that can be used to *, screen antibodies, proteins and enzymes for *' interactions with histones and their post- translational modifications. Each array con- ), tains 384 different histone modification com- )' binations in duplicate. Modifications include (, acetylation, methylation, phosphorylation If[Y_\_Y_jo\WYjeh (' and citrullination on the N-terminal tails of histones H2A, H2B, H3 and H4. , ' ?*B0d\*?*B).d\*?*I)d\)j?*I/d\)X?*I/d\)j?*B+XZ ?*B+d\*?*B+d\)?*B+d\(?*I)d\)X This unique histone array contains up to four modifications per 19mer peptide to study not only individual modifications, but also to Figure 2: ECL detection and graphical analysis of the cross-reactivity of Histone H3 trimethyl Lys9 antibody. Active Motif’s Histone H3 trimethyl Lys9 (H3K9me3) pAb (Catalog No. 39161) was used at a 1:2,000 dilution on the determine if neighboring modifications alter MODified Histone Peptide Array. Anti-rabbit HRP secondary antibody was used at a 1:2,500 dilution, followed by ECL site recognition and binding. The MODified detection and image capture with a CCD camera. Active Motif’s Array Analyse Software was used to analyze spot Histone Peptide Array can be used to screen intensity and generate a graphical analysis of decreasing specificity factors, which is the ratio of the average intensity of all spots containing H3K9me3 divided by the average intensity of all spots not containing H3K9me3. The results antibodies for cross-reactivity or to study show this antibody is very specific for histone H3 trimethyl Lys9, with little cross-reactivity for other modifications. protein and enzyme interactions (Figure 3). The simple array protocol works like a Advantages Free Software for Analysis Western blot. Either ECL-based or colorimet- • Histone specific – unique array panel Active Motif’s Array Analyse Software is ric detection systems can be used. The image tests for specific histone modifications a free program designed for use with the is captured using film or a CCD camera; no • Study neighboring effects – each MODified Histone Peptide Arrays. This PC special equipment is needed. peptide contains up to four modifica- compatible software will analyze the spot intensities from the MODified array and gen- The MODified Histone Peptide Arrays are tion combinations, enabling analysis of the effects of neighboring modifications erate a graphical analysis of the histone mod- available individually, or in packs of five. For ification interactions (Figure 2). Information a complete solution, the MODified Array • Detects like a Western blot – fast about spot intensity, averages and errors can Labeling Kit contains the necessary buffers and easy to use; works with either be saved in Excel-compatible files. For added and reagents for ECL-based detection. ECL-based or colorimetric detection convenience, up to three individual modifica- To learn more about the MODified™ Histone Peptide Arrays, or to download the free tions can be displayed in superposition to Array Analyse Software, please visit www.activemotif.com/modified. the experimental data, enabling better visualization of neighboring effects. Histone Antibodies Active Motif offers a variety of antibodies • Immunogen selection – ensures the To see our extensive list of histone to histones and biologically relevant histone antibody recognizes the modification of modification antibodies, including quality modifications. Each antibody has been interest, and does not cross-react with control data for IF, Western blot, ChIP or rigorously tested and validated for use in related proteins MODified Histone Peptide Array, please important applications such as immunofluo- • Specificity screening – every antibody visit www.activemotif.com/histoneabs. rescence (IF), Western blotting and chromatin must have a greater than 25-fold immunoprecipitation (ChIP). Our years of selectivity for the desired modification To learn about our fluorescent antibodies expertise in antibody development ensures for IF, or our antibody labeling kits, visit • Application validation – important that only the highest quality antibodies are www.activemotif.com/chromeo. applications are tested, giving you confi- offered for use in your research. dence when using them in your research *CelluSpots™ arrays are manufactured under license by INTAVIS Bioanalytical Instruments AG and sold through Active Motif as MODified™ Histone Peptide Arrays North America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638 Histone Analysis – tools designed to help unravel the histone code Histone Modifying Enzymes Histones are subject to a variety of post- These modifying enzymes can be used in 7 translational modifications that influence conjunction with Active Motif’s antibodies, a number of nuclear processes. To better recombinant histones, ELISAs and MODified™ investigate some of the complex functional Histone Peptide Arrays (Figure 3). questions about chromatin-associated pro- teins, nucleosome remodeling, transcriptional Figure 3: Images of ECL detection of MODified 8 regulation, replication and DNA repair, Active Histone Peptide Arrays treated with G9a. MODified Histone Peptide Arrays were treated with Motif offers a range of histone modifying A) 25 µM G9a methyltransferase (Catalog No. 31327), enzymes for the following protein types: B) 25 µM G9a mutant H904K (Catalog No. 31328), or • Methyltransferases C) no enzyme control, overnight in the presence of 1 mM AdoMet. The arrays were detected using a 9 • Demethylases Histone H3 dimethyl Lys9 antibody. Novel methyla-
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