Anti–H4K5,8,12ac polyclonal antibody

Catalog #

25283

$415 *

Size: 50 µg

Qty

Bulk/Custom

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*US Pricing only. For international pricing, please contact your local distributor.

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Description

Polyclonal antibody raised in rabbit against the region of histone H4 containing the acetylated lysines 5, 8 and 12 (H4K5,8,12ac), 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/66 µl

Immunogen

synthetic peptide

Background

Acetylation of histone H4 is associated with active genes.

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 H4K5,8,12ac
ChIP assays were performed using human K562 cells, the antibody against H4K5,8,12ac (Cat. # 25283) and optimized PCR primer sets for qPCR. ChIP was performed 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 analyzed. IgG (1 µg/IP) was used as negative IP control. QPCR was performed with primers for promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region 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 H4K5,8,12ac
ChIP was performed with 0.5 µg of the antibody against H4K5,8,12ac (Cat. # 25283) on sheared chromatin from 100,000 K562 cells. 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 signal distribution along the complete length of chromosome 2 (figure 2A) and a zoomin to a 600 kb region (figure 2B). Figure 2C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls.

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 H4K5,8,12ac (Cat. # 25283) in antigen coated wells. 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 1:14,500.

Cross reactivity tests using the antibody directed against H4K5,8,12ac
To test the cross reactivity of the antibody against H4K5,8,12ac (Cat. # 25283), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. 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 analysis using the antibody directed against H4K5,8,12ac
Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the antibody against H4K5,8,12ac (Cat. # 25283). 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 using the antibody directed against H4K5,8,12ac
HeLa cells were stained with the antibody against H4K5,8,12ac (Cat. # 25283) 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. Figure 6A: cells were immunofluorescently labeled with the H4K5,8,12ac 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. Figure 6B, C, D and E: staining of the cells with the H4K5,8,12ac antibody after incubation of the antibody with 10 ng/µl of the following blocking peptides: H4K5,8,12 unmodified (figure 6B), H4K5,8,12ac (figure 6C), H2A.ZK5,7,11ac (figure 6D) and H4K5,8,12,16ac (figure 6E).

Applications

ChIP/ChIP – seq (0.5 – 1 µg/IP)
ELISA (1:1000)
DB (1:20,000)
WB (1:1000)
IF (1:500)

Shipping Temperature

-20°C or-80°C

Species Reactivity

Human, mouse, broad range

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–H4K5,8,12ac polyclonal antibody

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