Anti–H3K9me3S10p polyclonal antibody

Catalog #

25273

$415 *

Size: 100 µl

Qty

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Description

Polyclonal antibody raised in rabbit against histone H3 containing trimethylated lysine 9 and the phosphorylated serine 10 (H3K9me3S10p), using a KLH–conjugated synthetic peptide.

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Product Info

Storage and Usage

Citations

Clonality

polyclonal

Host Species/Expression System

Rabbit

Purification

Whole serum

Format

aqueous solution

Formulation

Whole antiserum from rabbit containing 0.05% azide

Concentration

100 µl

Immunogen

synthetic peptide

Storage/Stability

Store at –80°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze/thaw cycles. Aliquots may be stored at –20°C for at least one month.

Assay Conditions

ChIP results obtained with the antibody directed against H3K9me3S10p
ChIP assays were performed using human HeLa cells treated with colcemid, the antibody against H3K9me3S10p (Cat. # 25273) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 10,000 cells per IP. A titration of the antibody consisting of 1, 5, and 10 µl per ChIP experiment was analyzed. Additionally, the same titration was analyzed after incubation of the antibody with 5 nmol of the corresponding blocking peptide for 1 hour at room temperature. IgG (5 µg/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active genes GAPDH and c-fos and for the heterochromatin marker Sat2 . Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Determination of the titer
To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against human H3K9me3S10p (Cat. # 25273). 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 1:87,000.

Cross reactivity test using the antibody directed against H3K9me3S10p
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K9me3S10p (Cat. # 25273) with peptides containing other modifications and unmodified sequences of histone H3. 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 1:1,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H3K9me3S10p
HeLa cells were treated with colcemid to block the cell cycle in metaphase and 15 µg of histone extracts of these cells were analyzed by Western blot with the antibody against H3K9me3S10p (Cat. # 25273) diluted 1:500 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. The result of the Western analysis with the antibody is shown in lane 2; lane 1 shows the same analysis after incubation of the antibody with 5 nmol of the corresponding blocking peptide for 1 hour at room temperature.

Immunofluorescence using the antibody directed against H3K9me3S10p
HeLa cells were stained with the antibody against H3K9me3S10p (Cat. # 25273) and with DAPI. Cells were fixed with 4% formaldehyde for 10 min. and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K9me3S10p 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

ChIP (5 µl/ChIP)
ELISA (1:1000 – 1:10,000)
DB (1:1000)
WB (1:500)
IF (1:500)

Shipping Temperature

-20°C or-80°C

Species Reactivity

Human

Cross Reactivity

No cross–reactivity with H3K9me(2 or 3), H3K9acS10p, or H3S10p. Very weak cross–reactivity with H3K9me1 at high concentrations.

Notes

The optimal antibody amount per IP should be determined by the end–user. We recommend testing 1–5 µg per IP

Warnings

Avoid freeze/thaw cycles

Scientific Category

Antibodies

Regulatory Status

Research Use Only

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Anti–H3K9me3S10p polyclonal antibody

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