Anti–H4K5ac polyclonal antibody

Catalog #

25284

$415 *

Size: 50 µg

Qty

Bulk/Custom

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Description

Polyclonal antibody raised in rabbit against the region of histone H4 containing the acetylated lysine 5 (H4K5ac), using a KLH–conjugated synthetic peptide.

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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/28 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 H4K5ac
ChIP assays were performed using human HeLa cells, the antibody against H4K5ac (Cat. # 25284) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 µg per ChIP experiment was analyzed. IgG (2 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 H4K5ac
ChIP was performed with 1 µg of the antibody against H4K5ac (Cat. # 25284) on sheared chromatin from 1 million HeLa 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 1 (figure 2A) and a zoomin to a 500 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. The position of the amplicon used for validating the QPCR results is shown 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 H4K5ac (Cat. # 25284) 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 1:8,900.

Cross reactivity tests using the antibody directed against H4K5ac
To test the cross reactivity of the antibody against H4K5ac (Cat. # 25284), 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 1:5,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H4K5ac
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 H4K5ac (Cat. # 25284). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.

Applications

ChIP/ChIP – seq (1 – 2 µg/IP)
ELISA (1:100 – 1:500)
DB (1:5000)
WB (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–H4K5ac polyclonal antibody

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