Diagenode

H4K5ac polyclonal antibody - Classic (sample size)

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410025-10
10 μg
$80.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against the region of histone H4 containing the acetylated lysine 5 (H4K5ac), using a KLH-conjugated synthetic peptide.

LotA1456D
Concentration1.80 µg/µl
Species reactivityHuman, mouse, wide range expected
TypePolyclonal
PurityAffinity purified
HostRabbit
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP * 1-2 μg/IP Fig 1, 2
ELISA 1:100-1:500 Fig 3
Dot Blotting 1:5,000 Fig 4
Western Blotting 1:500 Fig 5
Immunofluorescence 1:500 Fig 6

* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.

  • Validation data

    ChIP

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K5ac
    ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K5ac (cat. No. C15410025) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. AB-001-0024) on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

    ChIP-seq

    ChIP-seq

    ChIP-seq

    ChIP-seq

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K5ac
    ChIP was performed with 1 μg of the Diagenode antibody against H4K5ac (cat. No. C15410025) on sheared chromatin from 1 million HeLa cells using the “iDeal ChIP-seq” kit as described above. The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2 shows the signal distribution along the complete length of chromosome 1 (figure 2A) and a zoomin to a 500 kb region (figure 2B). Figure 2C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls. The position of the amplicon used for validating the QPCR results is shown with an arrow

    ELISA

    Figure 3. Determination of the antibody titer
    To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K5ac (cat. No. C15410025) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:8,900.

    Dot Blot

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K5ac
    To test the cross reactivity of the Diagenode antibody against H4K5ac (cat. No. C15410025), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:5,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

    Western Blot

    Figure 5. Western blot analysis using the Diagenode antibody directed against H4K5ac
    Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K5ac (cat. No. C15410025). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.

    Immunofluorescence

    Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K5ac
    HeLa cells were stained with the Diagenode antibody against H4K5ac (Cat. C15410025) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K5ac antibody (left) diluted 1:500 in blocking solution followed by an antirabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

  • Applications
    ELISA
    Enzyme-linked immunosorbent assay. Read more
    DB
    Dot blotting Read more
    WB
    Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more
    ChIP-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
    IF
    Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more
  • Documents
    Datasheet H4K5ac C15410025 DATASHEET
    Datasheet description
    Download
    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
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    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H4K5ac polyclonal antibody - Classic (sample size) (Diagenode Cat# C15410025-10 Lot# A1456D). Click here to copy to clipboard.

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Events

  • Workshop on Chromatin Proteomics
    Crete, Greece
    Oct 3-Oct 8, 2016
  • 2nd Annual Next Generation Sequencing Congress
    Boston, USA
    Oct 3-Oct 4, 2016
  • XXXIX Reunión anual de la Sociedad de Bioquimica y Biología Molecular de Chile
    Puerto Varas, Chile
    Sep 27-Sep 30, 2016
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