Diagenode

H3S10p polyclonal antibody - Classic (sample size)

RFX5-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410116-10
10 µg
$80.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against histone H3 containing the phosphorylated serine 10 (H3S10p), using a KLH-conjugated synthetic peptide.

LotA160-001234P
Concentration1.05 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 * 2 μg/ChIP Fig 1
ELISA 1:100 Fig 2
Dot Blotting 1:20,000 Fig 3
Western Blotting 1:1,000 Fig 4
Immunofluorescence 1:2,000 Fig 5

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

  • Validation Data

    ChIP

    ChIP

    Figure 1 ChIP results obtained with the Diagenode antibody directed against H3S10p
    Figure 1A: ChIP assays were performed using human HeLa cells treated with colcemid to block the cells in metaphase, the Diagenode antibody against H3S10p (Cat. No. pAb-116-050) and optimized PCR primer sets for qPCR. ChIP was performed with the “LowCell# ChIP” kit (Cat. No. kch-maglow-016) on sheared chromatin from 10,000 cells using 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 (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active genes c-fos (Cat. No. pp-1004-050) and RPL30. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). Figure 1B: ChIP was performed as described above using 2 μg of H3S10p antibody and sheared chromatin from 10,000 HeLa cells treated with colcemid (sample 1) or from 10,000 untreated cells (sample 2). QPCR was performed with primers for the promoter of the active genes c-fos and RPL30, and for the Sat2 satellite repeat region.

    ELISA

    Figure 2 Determination of the antibody titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3S10p (Cat. No. pAb-116-050) and the crude serum. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:5,200.

    Dot Blot

    Figure 3 Cross reactivity test with the Diagenode antibody directed against H3S10p
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3S10p (Cat. No. pAb-116-050) with peptides containing other modifications of histone H3 or the unmodified H3S10 sequence. One hundred to 0.2 pmol of the peptide containing the respective histone modification 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 modification of interest. Note that the antibody does not recognize the H3S10p modification if the neighboring K9 is acetylated or trimethylated.

    Western blot

    Figure 4 Western blot analysis using the Diagenode antibody directed against H3S10p
    HeLa cells were treated with colcemid, which blocks the cell cycle in metaphase and 15 μg of histone extracts of the cells were analysed by Western blot using the Diagenode antibody against H3S10p (Cat. No. pAb-116-050) 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 A

    Immunofluorescence figure B

    Immunofluorescence figure C

    Immunofluorescence figure D

    Figure 5 Immunofluorescence using the Diagenode antibody directed against H3S10p
    Human osteosarcoma (U2OS) cells were stained with the Diagenode antibody against H3S10p (Cat. No. pAb-116-050) and with DAPI. Cells were fixed with 3.7% formaldehyde in PBS for 20’ at RT, followed by a 20’ permeabilization with 0.5% Triton X-100 in PBS and blocked PBS/TX-100 containing 5% normal goat serum Figure 5A: cells were immunofluorescently labeled with the H3S10p antibody (left), diluted 1:2,000 in blocking buffer followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure 5B, C and D: staining of the cells with the H3S10p antibody after incubation of the antibody with 2 μM blocking peptide containing the unmodified H3S10 sequence, the phosphorylated H3S10 and the phosphorylated H3T11, 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
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H3S10p pAb-116-050 DATASHEET
    Datasheet description
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3S10p polyclonal antibody - Classic (sample size) (Diagenode Cat# C15410116-10 Lot# A160-001234P). Click here to copy to clipboard.

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