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Active Motif's Histone Analysis Brochure

Active Motif's Histone Analysis Brochure

Histone Analysis

Histone Analysis innovative tools designed to help unravel the

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 assembly. Histone Modification ELISAs Let Active Motif’s antibodies, enzymes and modification-specific Histone 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 , and at specific residues on the histone globular domain and the N-terminal tails have been shown to influence and regulate , 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 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 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. , Department of Biochemistry and Molecular , 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, 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 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 trimethyl Lys9 antibody. Active Motif’s Histone H3 trimethyl Lys9 () 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. 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 (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

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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 (Catalog No. 31327), enzymes for the following protein types: B) 25 µM G9a mutant H904K (Catalog No. 31328), or • C) no enzyme control, overnight in the presence of 1 mM AdoMet. The arrays were detected using a 9 • Histone H3 dimethyl Lys9 antibody. Novel methyla- tion sites were observed on the array treated with • Acetyltransferases wild-type G9a histone methyltransferase, showing the activity of this histone modifying enzyme on the • Deacetylases peptide substrates. To view our full listing of histone modifying enzymes and their associated technical data sheets, please visit www.activemotif.com/hismodenz.

Formaldehyde Detection of Histone Demethylase Activity

The fluorescent Histone Demethylase Assay The Histone Demethylase Assay Kit can 9ecfWh_iede\C[j^obWj[Z>_ijed[IkXijhWj[i is designed to detect the formaldehyde be used to analyze the overall conversion /' .' released from the reaction of specific efficiency of an LSD1 sample, or to screen -' demethylase 1 (LSD1, also known as KDM1) compounds that cause changes in histone ,' -) with a methylated substrate. The recom- demethylation activity. +' binant histone H3K4me2 substrate used in *' the assay mimics a native histone substrate, As shown in Figure 4, the LSD1 enzyme is able )' generating results that more closely resemble to more efficiently demethylate the included ('

HeLa Acid Extracts Active Motif’s HeLa acid extracts are a reli- or treated with chemicals known to affect To learn more, or to see a complete list able control for studying histone modifica- epigenetic events, such as sodium butyrate, of available extracts, please visit us at tions. Extracts are available either untreated, paclitaxel, etoposide and anacardic acid. www.activemotif.com/acidextract.

www.activemotif.com Recombinant Histone Proteins Active Motif is the first company to offer carefully controlled. Each methylation reac- recombinant histones with acetylation and tion is over 99% complete and is verified site-specific mono-, di- and trimethylation. by high-resolution mass spectrometry. The These recombinant histones can be used as recombinant histones are also analyzed by controls for histone antibodies, substrates dot blot to confirm identity (Figure 5). for histone modification enzymes, or to gen- erate chromatin in vitro, using Active Motif’s Acetylated histones are created with our Chromatin Assembly Kit (Catalog No. 53500). patent pending technology that enables us to acetylate the histone tail, without altering Recombinant methylated lysine residues are the native peptide bonds. The ability of the Figure 5: Dot blot of recombinant histones. One µg of unmodified, mono-, di- or trimethylated created using a patented approach in which acetylated and methylated histones to mimic recombinant proteins for H3K27, H3K36, H3K79 and an analog of methyl lysine is installed in the their native counterparts enables these sub- H4K20 were spotted onto a PVDF membrane and histone via chemical alkylation. This enables strates to yield more “natural” results than probed with Histone H3 dimethyl Lys79 pAb (Catalog the site and degree of methylation to be histone peptides. No. 39143) at a 1:1000 dilution. The dot blot confirms the identity of the recombinant protein. For a complete list of our over 20 unique recombinant histones, please visit us at www.activemotif.com/recombhis.

Histone Modification ELISAs

To better understand the effects of histone antibody against histone H3 and a detecting anti- >_ijed[>)cedec[j^obBoi(-;B?I7 (%) I\ZfdY`eXek?`jkfe\?* modifications on and body specific to the modification of interest. An I\ZfdY`eXek?`jkfe\?*dfefd\k_pcCpj). ( I\ZfdY`eXek?`jkfe\?*[`d\k_pcCpj). transcriptional regulation, Active Motif has HRP-conjugated secondary antibody and devel- I\ZfdY`eXek?`jkfe\?*ki`d\k_pcCpj). developed over 10 different assays for impor- oping solutions provide a colorimetric readout in '%/ tant histone modification sites, such as lysine less than 3 hours (Figure 6). '%- methylation at K4, K9 and K27 or serine '%+ phosphorylation at S10 and S28. These modi- Each kit includes validated modification '%)

specific controls. The included methylated >_ijed[>)cedec[j^obBoi(-E:*+&dc ' fication sites serve as key targets of histone ' (,%- *(%), -)%, (), ),' ,'' (''' H[YecX_dWdjfhej[_dd]%m[bb methyltransferase and histone demethylase recombinant histone proteins can be used to generate a standard curve, enabling Figure 6: Histone H3 monomethyl Lys27 specificity. enzymes, or act as markers for . Recombinant Histone H3, mono-, di- and trimethyl quantification of the amount of site- and Lys27 proteins were assayed from 15 ng - 1 µg per The Histone Modification ELISA Kits provide degree-specific methylated histone in each well using the Histone H3 monomethyl Lys27 ELISA. a sensitive method for detecting changes in sample. The acid extracts provided in the The results show the specificity of the assay for the the level of specific histone modifications phosphorylated ELISAs serve as a qualitative monomethyl modification. The monomethyl protein is included in the assay for sample quantification. from purified core histones, or histones control. The recombinant histones and acid isolated by acid extraction. These easy-to-use extracts are also available separately (see kits are sandwich ELISAs that utilize a capture above and on previous page).

To see an up-to-date list of the more than 10 Histone Modification ELISAs available, please visit www.activemotif.com/hiselisa.

Chromatin Assembly Design your own chromatin with Active components and incubate with DNA to generate The Chromatin Assembly Kit is an ATP-dependent Motif’s Chromatin Assembly Kit. Using either assembled chromatin that functions in a context method that utilizes purified recombinant human purified core histones, or Active Motif’s recom- that closely resembles in vivo chromatin. chromatin assembly complex ACF and histone binant histones, combine histones with the kit NAP-1 with core histones for in vitro assembly of extended, regularly ordered, periodic Product Format Catalog No. arrays of . For details, please visit Chromatin Assembly Kit 10 rxns 53500 www.activemotif.com/chromassembly.

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Histone Acetyltransferase (HAT) Activity The Histone Acetyltransferase (HAT) Assay Kit the provided acetyl-CoA to generate an is a quick and sensitive method to determine acetylated peptide and CoA-SH. After the activity of your own source of purified stopping the reaction with stop solution, a histone acetyltransferases, or to screen for developer is added that reacts with the free potential inhibitors of HAT activity. sulfhydryl groups on the CoA-SH to give a fluorescent signal (Figure 7). This fluorescent 96-well plate assay includes N-terminal histone H3 and H4 substrate A standard curve can be generated with peptides for screening HAT enzymes and a either b-mercaptoethanol or acetyl-CoA in positive control p300 catalytic domain order to relate the fluorescence of your HAT Figure 7: HAT inhibitor effects on p300 activity. protein to screen inhibitors. HATs will to pmol/min/µg specific activity. The HAT activity of 50 ng p300 catalytic domain was catalyze the transfer of acetyl groups from assayed using 50 µM acetyl-CoA and either 50 µM Histone H3 or peptides. The addition of 15 µM anacardic acid or 25 µM garcinol inhibited the Product Format Catalog No. HAT activity down to background levels. The back- HAT Assay Kit (Fluorescent) 1 x 96 rxns 56100 ground signal indicates the level of autoacetylation Recombinant p300 protein, catalytic domain 5 µg 31205 present from the p300 acetyltransferase.

Histone Deacetylase (HDAC) Activity To measure HDAC activity in your nuclear cofactor to the assay.) Once the substrate 50000 extracts, immunoprecipitates, column frac- is deacetylated, the lysine reacts with the Untreated tions or purified proteins, Active Motif offers developing solution and releases either a 40000 your choice of fluorescent or colorimetric chromophore or a fluorophore, which is then 30000 detection. Both HDAC kits utilize a short measured (Figure 8). A deacetylated assay 20000 peptide substrate containing an acetylated standard is provided in each kit to enable Intensity Fluorescence lysine residue that can be deacetylated by calculation of HDAC activity in pmol/min/mg. 10000 Class I, II and IV HDAC enzymes. (Class III & Additionally, the HDAC Assay Kits can be 0 10 52.5 1.25 0.625 0.312 0.156 0 Sirtuin HDACs require the addition of NAD+ used to screen inhibitor compounds. HeLa Nuclear Extract (µg) Figure 8: Fluorescent HDAC assay results. Product Format Catalog No. HeLa nuclear extracts were assayed for HDAC activity in duplicate from 0 to 10 µg per well in the pres- HDAC Assay Kit (Fluorescent) 1 x 96 rxns 56200 ence (copper line) or absence (purple line) of 1 µM HDAC Assay Kit (Colorimetric) 1 x 96 rxns 56210 Trichostatin A inhibitor.

Histone Binding Assay to Identify Chromatin-modifying Proteins Active Motif’s HiLite™ Binding Assay is an tion states, which in turn enables fast and calibration dye and five 96-well half area innovative tool for identifying chromatin- efficient inhibitor screening studies. black polystyrene plates. When the protein modifying proteins that bind to the histone of interest binds the modified peptide, it tails of methylated H3K9 and H3K27. This Each kit contains 8 fluorescently labeled slows the rotation of the peptide, causing fast, homogeneous assay uses fluorescence peptides that correspond to unmodified, the amount of polarized light that is emit- polarization (FP) to measure the affinity of mono-, di- and trimethylated histone H3 ted to be greater than an unbound peptide. the binding interactions between your pro- lysine 9 and lysine 27 regions, as well as a This provides a quantitative measure of the tein of interest and specific histone methyla- positive control HP1 protein, assay buffer, histone binding protein’s affinity for the peptide’s histone modification. Product Format Catalog No. HiLite™ Histone H3 Methyl-Lys9 / Lys27 1 kit 57001 To learn more about how this FP assay Binding Assay works, visit www.activemotif.com/hilite.

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