Diagenode

H3K27me3S28p polyclonal antibody - Classic

RFX5-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15310091
(CS-091-100)
100 µl
$295.00
  Bulk order

Polyclonal antibody raised in rabbit against histone H3 containing the trimethylated lysine 27 and the phosphorylated serine 28 (H3K27me3S28p), using a KLH-conjugated synthetic peptide.

LotA155-001
Concentrationnot determined
Species reactivityHuman
TypePolyclonal
PurityWhole antiserum
HostRabbit
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP * 1 μl/ChIP Fig 1
ELISA 1:100 - 1:500 Fig 2
Dot Blotting 1:20,000 Fig 3
Western Blotting 1:250 - 1:500 Fig 4, Fig 6
Immunofluorescence 1:200 Fig 5
Immunoprecipitation 5 μl/IP Fig 6
* Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 μl per IP.
  • Validation Data

    ChIP

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p
    ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

    ELISA

    Figure 2. Determination of the titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300.

    Dot Blot

    Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest.

    Western Blot

    Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p
    Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature.

    Immunofluorescence

    Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p
    Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells.

    Immunoprecipitation

    Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p
    HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively.

  • 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
    IP
    Immunoprecipitation Read more
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H3K27me3S28p CS-091-100 DATASHEET
    Datasheet description
    Download
  • Publications

    How to properly cite this product in your work

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