Diagenode

H2A.Zac polyclonal antibody - Premium (sample size)

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410202-10
10 µg
$80.00
  Bulk order
Other format

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

LotA1738P
Concentration1,09 µg/µl
Species reactivityHuman, mouse, other (wide range)
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/IP Fig 1, 2
ELISA 1:500 Fig 3
Dot Blotting 1:20,000 Fig 4
Western Blotting 1:1,000 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 0.5-5 μg per IP.
  • Validation Data

    ChIP figure A

    ChIP figure B

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H2A.Zac
    Figure 1A ChIP assays were performed using human HeLa cells, the Diagenode antibody against H2A.Zac (Cat. No. C15410202) and optimized PCR primer pairs for qPCR. ChIP was performed with the ““Auto Histone ChIP-seq” kit (Cat. No. AB-Auto02-A100) on the IP-Star automated system, using sheared chromatin from 1,000,000 cells. A titration consisting of 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Figure 1B ChIP assays were performed using human K562 cells, the Diagenode antibody against H2A.Zac (Cat. No. C15410202) 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. Quantitative PCR was performed with primers specific for the promoter of the active genes CCT5 and EIF4A2, used as positive controls, and for the coding region of the inactive MYT1 gene and the Sat2 satellite repeat, 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 figure A

    ChIP-seq figure B

    ChIP-seq figure C

    ChIP-seq figure D

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H2A.Zac
    ChIP was performed on sheared chromatin from 100,000 K562 cells using 1 μg the Diagenode antibody against H2A.Zac (Cat. No. C15410202) with the “iDeal ChIP-seq” kit (Cat. No. AB-001-0024) as described above. 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 2 shows the peak distribution along the complete sequence and a 1.5 Mb region of the X-chromosome (figure 2A and B) and in two regions surrounding the EIF4A2 and CCT5 positive control genes, respectively (figure 2C and D).

    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 against H2A.Zac (Cat. No. C15410202). 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 antibody was estimated to be 1:56,600.

    Dot blot

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H2A.Zac
    To test the cross reactivity of the Diagenode antibody against H2A.Zac (Cat. No. C15410202), a Dot Blot analysis was performed with peptides containing different multiple acetylations and the unmodified H2A.Z. 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 H2A.Zac
    Western blot was performed on whole cell extracts (25 μg, lane 1) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 2, 3, 4 and 5, respectively) using the Diagenode antibody against H2A.Zac (Cat. No. C15410202). 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

    Figure 6. Immunofluorescence using the Diagenode antibody directed against H2A.Zac
    HeLa cells were stained with the Diagenode antibody against H2A.Zac (Cat. No. C15410202) 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 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.

  • Background

    Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Acetylation of the histone H2A variant H2A.Z is associated with the promoters of active genes.

  • 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-qPCR (ab)
    Read more
  • Documents
    Datasheet H2AZac C15410202 DATASHEET
    Datasheet description
    Download
  • Publications

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  • Workshop on Chromatin Proteomics
    Crete, Greece
    Oct 3-Oct 8, 2016
  • 2nd Annual Next Generation Sequencing Congress
    Boston, USA
    Oct 3-Oct 4, 2016
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