Diagenode

H4K5,8,12ac polyclonal antibody - Classic (sample size)

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410021-10
10 μg
$80.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against the region of histone H4 containing the acetylated lysines 5, 8 and 12 (H4K5,8,12ac), using a KLH-conjugated synthetic peptide.

LotA2022P
Concentration0.76 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* 0.5 - 1 μg per IP Fig 1, 2
ELISA 1:1,000 Fig 3
Dot blotting 1:20,000 Fig 4
Western blotting 1:1,000 Fig 5
IF 1:500 Fig 6

* Please note that the optimal antibody amount per ChIP should be determined by the end-user. We recommend testing 0.5-5 μg per IP.

  • Validation Data

    ChIP

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K5,8,12ac.
    ChIP assays were performed using human K562 cells, the Diagenode antibody against H4K5,8,12ac (cat. No. C15410021) 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 100,000 cells. A titration of the antibody consisting of 0.2, 0.5, 1 and 2 μg per ChIP experiment was analysed. IgG (1 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 H4K5,8,12ac.
    ChIP was performed with 0.5 μg of the Diagenode antibody against H4K5,8,12ac (cat. No. C15410021) on sheared chromatin from 100,000 K562 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 2 (figure 2A) and a zoomin to a 600 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.

    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 H4K5,8,12ac (cat. No. C15410021) 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 1:14,500.

    Dot Blot

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K5,8,12ac.
    To test the cross reactivity of the Diagenode antibody against H4K5,8,12ac (cat. No. C15410021), 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 1:20,000. 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 H4K5,8,12ac.
    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 H4K5,8,12ac (cat. No. C15410021). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.

    Immunofluorescence

    Immunofluorescence

    Immunofluorescence

    Immunofluorescence

    Immunofluorescence

    Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K5,8,12ac.
    HeLa cells were stained with the Diagenode antibody against H4K5,8,12ac (cat. No. C15410021) 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. Figure 6A: cells were immunofluorescently labeled with the H4K5,8,12ac antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit 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. Figure 6B, C, D and E: staining of the cells with the H4K5,8,12ac antibody after incubation of the antibody with 10 ng/μl of the following blocking peptides: H4K5,8,12 unmodified (figure 6B), H4K5,8,12ac (figure 6C), H2A.ZK5,7,11ac (figure 6D) and H4K5,8,12,16ac (figure 6E).

  • 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
    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
    ChIP-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H4K5812ac C15410021 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...
    Download
    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: H4K5,8,12ac polyclonal antibody - Classic (sample size) (Diagenode Cat# C15410021-10 Lot# A2022P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

  • Related products

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
 See all events

Twitter feed

News

 See all news