Anti–H3K79me2 polyclonal antibody

Catalog #

25262

$415 *

Size: 50 µg

Qty

Bulk/Custom

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Description

Polyclonal antibody raised in rabbit against the region of histone H3 containing the dimethylated lysine 79 (H3K79me2), using a KLH–conjugated synthetic peptide

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Product Data Gallery

Product Info

Storage and Usage

Citations

Clonality

polyclonal

Host Species/Expression System

Rabbit

Purification

Affinity purified

Format

Aqueous buffer solution

Formulation

PBS containing 0.05% azide and 0.05% ProClin 300

Concentration

50 µg/46 µl

Immunogen

synthetic peptide

Background

Methylation of H3K79 is usually catalyzed by the methyltransferase Dot1L and is associated with gene transcription.

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 H3K79me2
ChIP assays were performed using human HeLa cells, the antibody against H3K79me2 (Cat. # 25262) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million 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. Quantitative PCR was performed with primers specific for the coding regions of the active genes ACTB and ALDOA, used as positive controls, and for exon 2 of the inactive myoglobin (MB) 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 results obtained with the antibody directed against H3K79me2
ChIP was performed with 1 µg of the antibody against H3K79me2 (Cat. # 25262) as described above and the immunoprecipitated DNA was subsequently analyzed on 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 Mb region of the X-chromosome (figure 2A and B), in a 100 kb region surrounding the ACTB positive control gene (figure 2C) and in a 20 kb region surrounding the ALDOA gene (figure 2D). The position of the amplicon used for validating the ALDOA enrichment is indicated with an arrow.

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against human H3K79me2 (Cat. # 25262), crude serum and flow-through. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:8,000.

Cross reactivity tests using the antibody directed against H3K79me2
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K79me2 (Cat. # 25262) with peptides containing other modifications and unmodified sequences of histone H3. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:50,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H3K79me2
Histone extracts (15 µg) from HeLa cells were analyzed by Western blot using the antibody against H3K79me2 (Cat. # 25262) 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 using the antibody directed against H3K79me2
HeLa cells were stained with the antibody against H3K79me2 (Cat. # 25262) 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 H3K79me2 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/ChIP – seq (5 µg/ChIP)
ELISA (1:100)
DB (1:50,000)
WB (1:1000)
IF (1:500)

Shipping Temperature

-20°C or-80°C

Species Reactivity

Human

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–H3K79me2 polyclonal antibody

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