

HA Tag CUTANA™ CUT&RUN Antibody
{"url":"https://www.epicypher.com/products/antibodies/cutana-cut-run-compatible-antibodies/ha-tag-cutana-cut-and-run-antibody","add_this":[{"service":"facebook","annotation":""},{"service":"email","annotation":""},{"service":"print","annotation":""},{"service":"twitter","annotation":""},{"service":"linkedin","annotation":""}],"gtin":null,"id":"875","bulk_discount_rates":[],"can_purchase":true,"meta_description":"Rabbit polyclonal HA Tag antibody validated for CUT&RUN and WB. Rigorously tested for reliable and robust performance in CUT&RUN assays.","category":["Antibodies/CUTANA™ CUT&RUN Antibodies","Antibodies/CUTANA™ CUT&RUN Antibodies/CUT&RUN Antibodies - Chromatin-Associated Proteins","Epigenetics Kits and Reagents/CUTANA™ ChIC / CUT&RUN Assays"],"AddThisServiceButtonMeta":"","main_image":{"data":"https://cdn11.bigcommerce.com/s-y9o92/images/stencil/{:size}/products/875/937/ha-tag-cutana-cutandrun-antibody__43540.1645734529.jpg?c=2","alt":"HA Tag CUTANA CUTandRUN Antibody"},"add_to_wishlist_url":"/wishlist.php?action=add&product_id=875","shipping":{"calculated":true},"num_reviews":0,"weight":"0.00 LBS","custom_fields":[{"id":"909","name":"Pack Size","value":"100 µg"}],"sku":"13-2010","description":"<div class=\"product-general-info\">\n <ul class=\"product-general-info__list-left\">\n <li class=\"product-general-info__list-item\">\n <strong>Type: </strong>Polyclonal\n </li>\n <li class=\"product-general-info__list-item\">\n <strong>Host: </strong>Rabbit\n </li>\n <li class=\"product-general-info__list-item\">\n <strong>Applications: </strong>CUT&RUN, WB\n </li>\n </ul>\n <ul class=\"product-general-info__list-right\">\n <li class=\"product-general-info__list-item\">\n <strong>Reactivity: </strong>HA Epitope (YPYDVPDYA)\n </li>\n <li class=\"product-general-info__list-item\">\n <strong>Format: </strong>Antigen affinity-purified\n </li>\n <li class=\"product-general-info__list-item\">\n <strong>Target Size: </strong>N/A\n </li>\n </ul>\n</div>\n<div class=\"service_accordion product-droppdown\">\n <div class=\"container\">\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel current\">\n <h3 class=\"sub-title1\">Description</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description-specific\">\n <p>\n This antibody meets EpiCypher’s \"CUTANA Compatible\" criteria for\n performance in Cleavage Under Targets and Release Using Nuclease\n (CUT&RUN) and/or Cleavage Under Targets and Tagmentation (CUT&Tag)\n approaches to genomic mapping. Every lot of a CUTANA Compatible\n antibody is tested in the indicated approach using\n <a href=\"/protocols\">EpiCypher optimized protocols</a> and\n determined to yield peaks that show a genomic distribution pattern\n consistent with reported function(s) of the target. HA antibody is\n useful for studies utilizing HA-tagged target proteins. HA Tag\n antibody produces CUT&RUN peaks (<strong>Figure 1</strong>) that\n overlap with GATA3 DNA-binding motifs (<strong>Figure 2</strong>) in\n breast cancer cells expressing 3xHA-tagged GATA3 transcription\n factor [1]*.\n </p>\n </div>\n </div>\n </div>\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel current\">\n <h3 class=\"sub-title1\">Validation Data</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description-specific\">\n <p>\n CUT&RUN was performed on 500k MDA-MB-231 cells stably expressing\n c-terminal 3xHA-tagged GATA3 [1]* with 0.5 µg of either HA Tag,\n H3K4me3 positive control (EpiCypher\n <a\n href=\"/products/antibodies/snap-chip-certified-antibodies/histone-h3k4me3-antibody-snap-chip-certified-cutana-cut-run-compatible\"\n >13-0041</a\n >), or IgG negative control (EpiCypher\n <a\n href=\"/products/nucleosomes/snap-cutana-spike-in-controls/cutana-rabbit-igg-cut-run-negative-control-antibody\"\n >13-0042</a\n >) antibodies using the CUTANA™ ChIC/CUT&RUN Kit v2.0 (EpiCypher\n <a\n href=\"/products/epigenetics-reagents-and-assays/cutana-chic-cut-and-run-kit\"\n >14-1048</a\n >). Library preparation was performed with 5 ng of DNA (or the total\n amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN\n Library Prep Kit (EpiCypher\n <a\n href=\"/products/epigenetics-reagents-and-assays/cutana-cut-and-run-library-prep-kit\"\n >14-1001/14-1002</a\n >). Both kit protocols were adapted for high throughput Tecan liquid\n handling. Libraries were run on an Illumina NextSeq2000 with\n paired-end sequencing (2x50 bp). Sample sequencing depth was 2.4\n million reads (IgG), 4.1 million reads (H3K4me3), and 8.6 million\n reads (HA Tag). Data were aligned to the hg19 genome using Bowtie2.\n Data were filtered to remove duplicates, multi-aligned reads, and\n blacklist regions.\n </p>\n <section class=\"image-picker\">\n <div class=\"image-picker__left\">\n <div\n class=\"image-picker__main-content_active image-picker__main-content\">\n <div class=\"image-picker__header-content\">\n <button class=\"image-picker__left-arrow\">\n <svg\n class=\"image-picker__svg-left\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M16.67 0l2.83 2.829-9.339 9.175 9.339 9.167-2.83 2.829-12.17-11.996z\" />\n </svg>\n </button>\n <a\n href=\"/content/images/products/antibodies/13-2010-HA-Tag-Heatmap.jpeg\"\n target=\"_new\"\n class=\"image-picker__main-image-link\"\n ><img loading=\"lazy\"\n alt=\"13-2010-HA-Tag-Heatmap\"\n src=\"/content/images/products/antibodies/13-2010-HA-Tag-Heatmap.jpeg\"\n class=\"image-picker__main-image\" />\n <span class=\"image-picker__main-image-caption\"\n >(Click to enlarge)</span\n ></a\n >\n <button class=\"image-picker__right-arrow\">\n <svg\n class=\"image-picker__svg-right\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M7.33 24l-2.83-2.829 9.339-9.175-9.339-9.167 2.83-2.829 12.17 11.996z\" />\n </svg>\n </button>\n </div>\n <p>\n <span class=\"image-picker__span-content\"\n ><strong>Figure 1: HA Tag peaks in CUT&RUN </strong><br />\n CUT&RUN was performed as described above. Peaks were called\n using MACS2. (<strong>A</strong>) Heatmaps show GATA3-3xHA\n peaks relative to IgG and H3K4me3 control antibodies in\n aligned rows ranked by intensity (top to bottom) and colored\n such that red indicates high localized enrichment and blue\n denotes background signal. (<strong>B</strong>) The number\n of GATA3-3xHA peaks that fall into distinct classes of\n functionally annotated genomic regions are shown.\n </span>\n </p>\n </div>\n <div class=\"image-picker__main-content\">\n <div class=\"image-picker__header-content\">\n <button class=\"image-picker__left-arrow\">\n <svg\n class=\"image-picker__svg-left\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M16.67 0l2.83 2.829-9.339 9.175 9.339 9.167-2.83 2.829-12.17-11.996z\" />\n </svg>\n </button>\n <a\n href=\"/content/images/products/antibodies/13-2010-HA-Tag-Motif.jpeg\"\n target=\"_new\"\n class=\"image-picker__main-image-link\"\n ><img loading=\"lazy\"\n alt=\"13-2010-HA-Tag-Motif\"\n src=\"/content/images/products/antibodies/13-2010-HA-Tag-Motif.jpeg\"\n class=\"image-picker__main-image\" />\n <span class=\"image-picker__main-image-caption\"\n >(Click to enlarge)</span\n ></a\n >\n <button class=\"image-picker__right-arrow\">\n <svg\n class=\"image-picker__svg-right\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M7.33 24l-2.83-2.829 9.339-9.175-9.339-9.167 2.83-2.829 12.17 11.996z\" />\n </svg>\n </button>\n </div>\n <p>\n <span class=\"image-picker__span-content\"\n ><strong\n >Figure 2: HA-tagged transcription factor binding motif\n analysis in CUT&RUN</strong\n ><br />\n (<strong>A</strong>) Homer analysis determined that the\n GATA3 consensus motif, represented as a sequence logo\n position weight matrix, was enriched under GATA3-3xHA\n CUT&RUN peaks. (<strong>B</strong>) The number of GATA3-3xHA\n peaks containing GATA3 consensus motifs from panel A is\n represented by a Venn Diagram. (<strong>C-D</strong>) Two\n representative loci show overlap of GATA3-3xHA peaks with\n the consensus motifs noted by tick marks beneath the tracks.\n </span>\n </p>\n </div>\n\n <div class=\"image-picker__main-content\">\n <div class=\"image-picker__header-content\">\n <button class=\"image-picker__left-arrow\">\n <svg\n class=\"image-picker__svg-left\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M16.67 0l2.83 2.829-9.339 9.175 9.339 9.167-2.83 2.829-12.17-11.996z\" />\n </svg>\n </button>\n <a\n href=\"/content/images/products/antibodies/13-2010-western-blot.jpeg\"\n target=\"_new\"\n class=\"image-picker__main-image-link\"\n ><img loading=\"lazy\"\n alt=\"13-2010-western-blot\"\n src=\"/content/images/products/antibodies/13-2010-western-blot.jpeg\"\n class=\"image-picker__main-image\" />\n <span class=\"image-picker__main-image-caption\"\n >(Click to enlarge)</span\n ></a\n >\n <button class=\"image-picker__right-arrow\">\n <svg\n class=\"image-picker__svg-right\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M7.33 24l-2.83-2.829 9.339-9.175-9.339-9.167 2.83-2.829 12.17 11.996z\" />\n </svg>\n </button>\n </div>\n <p>\n <span class=\"image-picker__span-content\"\n ><strong>Figure 3: Western blot data</strong><br />\n <em>E. coli</em> cells expressing a multi-tag fusion protein\n were used to prepare whole cell lysates. The indicated\n amounts (ng) of lysate were loaded onto a 4-20% SDS-PAGE gel\n and analyzed under standard western blot conditions using HA\n Tag antibody at a dilution of 1:25,000.\n </span>\n </p>\n </div>\n <div class=\"image-picker__main-content\">\n <div class=\"image-picker__header-content\">\n <button class=\"image-picker__left-arrow\">\n <svg\n class=\"image-picker__svg-left\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M16.67 0l2.83 2.829-9.339 9.175 9.339 9.167-2.83 2.829-12.17-11.996z\" />\n </svg>\n </button>\n <a\n href=\"/content/images/products/antibodies/13-2010-HA-Tag-ChAP-Spike-ins.jpeg\"\n target=\"_new\"\n class=\"image-picker__main-image-link\"\n ><img loading=\"lazy\"\n alt=\"13-2010-HA-Tag-ChAP-Spike-ins\"\n src=\"/content/images/products/antibodies/13-2010-HA-Tag-ChAP-Spike-ins.jpeg\"\n class=\"image-picker__main-image\" />\n <span class=\"image-picker__main-image-caption\"\n >(Click to enlarge)</span\n ></a\n >\n <button class=\"image-picker__right-arrow\">\n <svg\n class=\"image-picker__svg-right\"\n width=\"24\"\n height=\"24\"\n viewBox=\"0 0 24 24\">\n <path\n d=\"M7.33 24l-2.83-2.829 9.339-9.175-9.339-9.167 2.83-2.829 12.17 11.996z\" />\n </svg>\n </button>\n </div>\n <p>\n <span class=\"image-picker__span-content\"\n ><strong\n >Figure 4: Target-specific epitope cleavage of HA Tag\n antibody in CUT&RUN was determined using DNA-barcoded\n recombinant nucleosome spike-in controls</strong\n ><br />\n (<strong>A</strong>) A panel of recombinant nucleosomes was\n created where various epitope tags (3xTY1, 3xFLAG, 3xHA)\n were fused to the histone H3 tail. The fused nucleosomes and\n an unmodified control were immobilized to streptavidin beads\n (SA Bead) and spiked into CUT&RUN samples alongside ConA\n bead immobilized MDA-MB-231 cells expressing GATA3-3xHA\n (Figure 1). HA Tag antibody and pAG-MNase (EpiCypher\n <a\n href=\"https://www.epicypher.com/products/epigenetics-reagents-and-assays/cutana-pag-mnase-for-chic-cut-and-run-workflows\"\n >15-1016</a\n >) were then added to release antibody-bound nucleosomes\n into solution through pAG-MNase mediated cleavage of the\n linker DNA (light blue). This approach provided a defined\n experimental control to assess whether the HA Tag antibody\n selectively cleaved the target epitope with high specificity\n and minimal background. (<strong>B</strong>) CUT&RUN\n sequence reads were aligned to the unique DNA \"barcodes\"\n corresponding to each nucleosome in the spike-in panel. Data\n are expressed as the percent of reads recovered relative to\n the intended target (3xHA, set to 100%). This analysis\n confirms that the HA Tag antibody specifically liberated the\n target epitope-tagged nucleosome into solution.\n </span>\n </p>\n </div>\n </div>\n <aside class=\"image-picker__right\">\n <div class=\"image-picker__gallery\">\n <img loading=\"lazy\"\n alt=\"13-2010-HA-Tag-Heatmap\"\n src=\"/content/images/products/antibodies/13-2010-HA-Tag-Heatmap.jpeg\"\n width=\"200\"\n class=\"image-picker__side-image image-picker__side-image_active\"\n role=\"button\" />\n <img loading=\"lazy\"\n alt=\"13-2010-HA-Tag-Motif\"\n src=\"/content/images/products/antibodies/13-2010-HA-Tag-Motif.jpeg\"\n class=\"image-picker__side-image\"\n role=\"button\" />\n <img loading=\"lazy\"\n alt=\"13-2010-western-blot\"\n src=\"/content/images/products/antibodies/13-2010-western-blot.jpeg\"\n class=\"image-picker__side-image\"\n role=\"button\" />\n <img loading=\"lazy\"\n alt=\"13-2010-HA-Tag-ChAP-Spike-ins\"\n src=\"/content/images/products/antibodies/13-2010-HA-Tag-ChAP-Spike-ins.jpeg\"\n class=\"image-picker__side-image\"\n role=\"button\" />\n </div>\n </aside>\n </section>\n </div>\n </div>\n </div>\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel\">\n <h3 class=\"sub-title1\">Technical Information</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description\">\n <div class=\"product-tech-info\">\n <div class=\"product-tech-info__line-item\">\n <div class=\"product-tech-info__line-item-left\">\n <b>Immunogen</b>\n </div>\n <div class=\"product-tech-info__line-item-right\">\n A synthetic HA peptide (sequence: YPYDVPDYA)\n </div>\n </div>\n <div class=\"product-tech-info__line-item\">\n <div class=\"product-tech-info__line-item-left\">\n <b>Storage</b>\n </div>\n <div class=\"product-tech-info__line-item-right\">\n Stable for 1 year at 4°C from date of receipt\n </div>\n </div>\n <div class=\"product-tech-info__line-item\">\n <div class=\"product-tech-info__line-item-left\">\n <b>Formulation</b>\n </div>\n <div class=\"product-tech-info__line-item-right\">\n Antigen affinity-purified antibody in phosphate buffered saline\n (PBS), 0.09% sodium azide\n </div>\n </div>\n </div>\n </div>\n </div>\n </div>\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel\">\n <h3 class=\"sub-title1\">Recommended Dilution</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description\">\n <div class=\"product-tech-info\">\n <div class=\"product-tech-info__line-item\">\n <div class=\"product-tech-info__line-item-left\">\n <b>CUT&RUN:</b>\n </div>\n <div class=\"product-tech-info__line-item-right\">\n 0.5 µg per reaction\n </div>\n </div>\n <div class=\"product-tech-info__line-item\">\n <div class=\"product-tech-info__line-item-left\">\n <b>Western Blot (WB):</b>\n </div>\n <div class=\"product-tech-info__line-item-right\">\n 1:1,000 - 1:30,000\n </div>\n </div>\n </div>\n </div>\n </div>\n </div>\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel\">\n <h3 class=\"sub-title1\">References</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description\">\n <strong>Background References:</strong>\n <br />\n [1] Takaku et al. <em>Genome Biol.</em> (2016). PMID:\n <a\n href=\"https://pubmed.ncbi.nlm.nih.gov/26922637/\"\n title=\"GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler\"\n target=\"new\">\n 26922637</a\n >\n <br />\n <p>\n <em\n >*Thanks to Dr. Takaku (UND) for 3xFlag-GATA3-3xHA MDA-MB-231\n cells.</em\n >\n </p>\n </div>\n </div>\n </div>\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel\">\n <h3 class=\"sub-title1\">Documents & Resources</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description\">\n <div class=\"product-documents\">\n <a\n href=\"/content/documents/tds/13-2010.pdf\"\n target=\"_new\"\n class=\"product-documents__link\">\n <svg\n version=\"1.1\"\n id=\"Layer_1\"\n xmlns=\"http://www.w3.org/2000/svg\"\n xmlns:xlink=\"http://www.w3.org/1999/xlink\"\n x=\"0px\"\n y=\"0px\"\n viewBox=\"0 0 228 240\"\n style=\"enable-background: new 0 0 228 240\"\n xml:space=\"preserve\"\n 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d=\"M69.92,125.68h18.91c5.29,0,9.84,0.97,13.66,2.9c3.82,1.93,6.74,4.72,8.76,8.35\n c2.02,3.63,3.04,7.98,3.04,13.04c0,5.06-1,9.42-3,13.08c-2,3.66-4.91,6.45-8.73,8.38c-3.82,1.93-8.4,2.9-13.73,2.9H69.92V125.68z\n M88.07,165.63c10.35,0,15.52-5.22,15.52-15.66c0-10.4-5.17-15.59-15.52-15.59h-7.38v31.26H88.07z\" />\n <path\n class=\"product-documents__svg-pdf\"\n d=\"M122.57,125.68h32.84v8.49h-22.22v11.18h20.84v8.49h-20.84v20.49h-10.63V125.68z\" />\n </g>\n </svg>\n <span class=\"product-documents__info\">Technical Datasheet</span>\n </a>\n </div>\n </div>\n </div>\n </div>\n <div id=\"prodAccordion\">\n <div id=\"ProductDescription\" class=\"Block Panel\">\n <h3 class=\"sub-title1\">Additional Info</h3>\n <div\n class=\"ProductDescriptionContainer product-droppdown__section-description\">\n <p>\n This product is provided for commercial sale under license from\n Bethyl Laboratories, Inc.\n </p>\n </div>\n </div>\n </div>\n </div>\n</div>\n","tags":[],"warranty":"","price":{"without_tax":{"formatted":"$495.00","value":495,"currency":"USD"},"tax_label":"Sales Tax"},"detail_messages":"","availability":"","page_title":"HA Tag Antibody | CUTANA™ CUT&RUN Compatible","cart_url":"https://www.epicypher.com/cart.php","max_purchase_quantity":0,"mpn":null,"upc":null,"options":[],"related_products":[{"id":694,"sku":null,"name":"CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows","url":"https://www.epicypher.com/products/epigenetics-reagents-and-assays/cutana-pag-mnase-for-chic-cut-and-run-workflows","availability":"","rating":null,"brand":{"name":null},"category":["Epigenetics Kits and Reagents","Epigenetics Kits and Reagents/CUTANA™ ChIC / CUT&RUN Assays"],"summary":"\n \n \n Type: Nuclease\n \n \n Mol Wgt: 43.7 kDa\n \n \n \n ","image":{"data":"https://cdn11.bigcommerce.com/s-y9o92/images/stencil/{:size}/products/694/689/Screen_Shot_2020-02-12_at_11.01.55_AM__17144.1581530752.png?c=2","alt":"CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows"},"images":[{"data":"https://cdn11.bigcommerce.com/s-y9o92/images/stencil/{:size}/products/694/689/Screen_Shot_2020-02-12_at_11.01.55_AM__17144.1581530752.png?c=2","alt":"CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows"}],"date_added":"12th Aug 2019","pre_order":false,"show_cart_action":true,"has_options":true,"stock_level":null,"low_stock_level":null,"qty_in_cart":0,"custom_fields":[{"id":1174,"name":"Internal Comment","value":"Excess in bottom of Venom"},{"id":1175,"name":"Internal Comment","value":"Bulk in Psylocke"}],"num_reviews":null,"weight":{"formatted":"0.01 LBS","value":0.01},"demo":false,"price":{"without_tax":{"currency":"USD","formatted":"$315.00","value":315},"price_range":{"min":{"without_tax":{"currency":"USD","formatted":"$315.00","value":315},"tax_label":"Sales Tax"},"max":{"without_tax":{"currency":"USD","formatted":"$1,250.00","value":1250},"tax_label":"Sales Tax"}},"tax_label":"Sales 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- Type: Polyclonal
- Host: Rabbit
- Applications: CUT&RUN, WB
- Reactivity: HA Epitope (YPYDVPDYA)
- Format: Antigen affinity-purified
- Target Size: N/A
Description
This antibody meets EpiCypher’s "CUTANA Compatible" criteria for performance in Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and/or Cleavage Under Targets and Tagmentation (CUT&Tag) approaches to genomic mapping. Every lot of a CUTANA Compatible antibody is tested in the indicated approach using EpiCypher optimized protocols and determined to yield peaks that show a genomic distribution pattern consistent with reported function(s) of the target. HA antibody is useful for studies utilizing HA-tagged target proteins. HA Tag antibody produces CUT&RUN peaks (Figure 1) that overlap with GATA3 DNA-binding motifs (Figure 2) in breast cancer cells expressing 3xHA-tagged GATA3 transcription factor [1]*.
Validation Data
CUT&RUN was performed on 500k MDA-MB-231 cells stably expressing c-terminal 3xHA-tagged GATA3 [1]* with 0.5 µg of either HA Tag, H3K4me3 positive control (EpiCypher 13-0041), or IgG negative control (EpiCypher 13-0042) antibodies using the CUTANA™ ChIC/CUT&RUN Kit v2.0 (EpiCypher 14-1048). Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Both kit protocols were adapted for high throughput Tecan liquid handling. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 2.4 million reads (IgG), 4.1 million reads (H3K4me3), and 8.6 million reads (HA Tag). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and blacklist regions.
Figure 1: HA Tag peaks in CUT&RUN
CUT&RUN was performed as described above. Peaks were called
using MACS2. (A) Heatmaps show GATA3-3xHA
peaks relative to IgG and H3K4me3 control antibodies in
aligned rows ranked by intensity (top to bottom) and colored
such that red indicates high localized enrichment and blue
denotes background signal. (B) The number
of GATA3-3xHA peaks that fall into distinct classes of
functionally annotated genomic regions are shown.
Figure 2: HA-tagged transcription factor binding motif
analysis in CUT&RUN
(A) Homer analysis determined that the
GATA3 consensus motif, represented as a sequence logo
position weight matrix, was enriched under GATA3-3xHA
CUT&RUN peaks. (B) The number of GATA3-3xHA
peaks containing GATA3 consensus motifs from panel A is
represented by a Venn Diagram. (C-D) Two
representative loci show overlap of GATA3-3xHA peaks with
the consensus motifs noted by tick marks beneath the tracks.
Figure 3: Western blot data
E. coli cells expressing a multi-tag fusion protein
were used to prepare whole cell lysates. The indicated
amounts (ng) of lysate were loaded onto a 4-20% SDS-PAGE gel
and analyzed under standard western blot conditions using HA
Tag antibody at a dilution of 1:25,000.
Figure 4: Target-specific epitope cleavage of HA Tag
antibody in CUT&RUN was determined using DNA-barcoded
recombinant nucleosome spike-in controls
(A) A panel of recombinant nucleosomes was
created where various epitope tags (3xTY1, 3xFLAG, 3xHA)
were fused to the histone H3 tail. The fused nucleosomes and
an unmodified control were immobilized to streptavidin beads
(SA Bead) and spiked into CUT&RUN samples alongside ConA
bead immobilized MDA-MB-231 cells expressing GATA3-3xHA
(Figure 1). HA Tag antibody and pAG-MNase (EpiCypher
15-1016) were then added to release antibody-bound nucleosomes
into solution through pAG-MNase mediated cleavage of the
linker DNA (light blue). This approach provided a defined
experimental control to assess whether the HA Tag antibody
selectively cleaved the target epitope with high specificity
and minimal background. (B) CUT&RUN
sequence reads were aligned to the unique DNA "barcodes"
corresponding to each nucleosome in the spike-in panel. Data
are expressed as the percent of reads recovered relative to
the intended target (3xHA, set to 100%). This analysis
confirms that the HA Tag antibody specifically liberated the
target epitope-tagged nucleosome into solution.
Technical Information
Recommended Dilution
References
[1] Takaku et al. Genome Biol. (2016). PMID: 26922637
*Thanks to Dr. Takaku (UND) for 3xFlag-GATA3-3xHA MDA-MB-231 cells.
Documents & Resources
Additional Info
This product is provided for commercial sale under license from Bethyl Laboratories, Inc.