Diagenode

H2A.Zac polyclonal antibody - Classic

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410173
(pAb-173-050)
50 μg/72 μl
$295.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against histone H2A.Z acetylated at lysines 5, 7 and 11, using a KLH-conjugated synthetic peptide.

LotA0405-001P
Concentration0.7 µg/µl
Species reactivityHuman
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 μg/ChIP Fig 1, 2
ELISA 1:200 Fig 3
Dot Blotting 1:20,000 Fig 4
Immunofluorescence 1:500 Fig 5
* 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 H2A.Zac
    ChIP assays were performed using HeLa cells, the Diagenode antibody against H2A.Zac (Cat. No. pAb-173-050) and optimized primer pairs for qPCR. ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using the “Auto Histone ChIP-seq” kit (Cat. No. AB-Auto02-A100) on the SX-8G IP-Star automated system. 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 using primers specific for the promoters of the ACTB and EIF4A2 genes, used as positive control targets and for the coding region of the MYT1 gene, used as a negative control target. Figure 1 shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of H2A.Z is present at active promoters.

    ChIP-seq figure A

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H2A.Zac
    ChIP was performed with 1 μg of the Diagenode antibody against H2A.Zac (Cat. No. pAb-173-050) on sheared chromatin from 1 million HeLaS3 cells using the “iDeal ChIP-seq” kit (Cat. No. AB-001-0024). IgG (2 μg/IP) was used as a negative IP control. The IP’d DNA was analysed by QPCR as described above (figure 1A). The IP’d DNA was subsequently analysed with 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 1 shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure 1B and C) and in 100 kb regions surrounding the EIF4A2, ACTB and GAPDH genes (figure 1D, E and F). These results clearly show an enrichment of the H2A.Z acetylation at the promoters of active genes.

    ChIP-seq figure B

    ChIP-seq figure C

    ChIP-seq figure D

    ChIP-seq figure E

    ChIP-seq figure F

    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 H2A.Zac (Cat. No. pAb-173-050), crude serum and flow through in antigen coated wells. The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the purified antibody was estimated to be 1:8,800.

    Dot Blot

    Figure 4. Cross reactivity test using the Diagenode antibody directed against H2A.Zac
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H2A.Zac (Cat. No. pAb-173- 050) with peptides containing other histone acetylations and the unmodified H2A.Z sequence. 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.

    Immunofluorescence

    Figure 5. Immunofluorescence using the Diagenode antibody directed against H2A.Zac
    HeLa cells were stained with the Diagenode antibody against H2A.Zac (cat. No. pAb-173-050) 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 labelled with the H2A.Zac 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.

  • Applications
    ELISA
    Enzyme-linked immunosorbent assay. Read more
    DB
    Dot blotting 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 H2AZac pAb-173-050 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...
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  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H2A.Zac polyclonal antibody - Classic (Diagenode Cat# C15410173 Lot# A0405-001P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.
    Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC
    BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the unde...

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