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Applications: ChIP/ChIP - seq (1 - 2 µg/ChIP)
ELISA (1:100)
DB (1:25000)
WB (1:1000)
IF (1:500)
Assay Conditions: ChIP results obtained with the antibody directed against H3K27ac
ChIP assays were performed using HeLa cells, the antibody against H3K27ac (Cat. No. 25239) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the promoter of the active ACTB and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and TSH2B genes, 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). These results are in accordance with the observation that H3K27 acetylation is enriched at the promoters of active genes.ChIP-seq results obtained with the antibody directed against H3K27ac
ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 2 µg of the antibody against H3K27ac (Cat. No. 25239) as described above. Quantitative PCR was performed with primers for the promoter of the active ACTB and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and MB genes, used as negative controls (Figure 2A). The immunoprecipitated DNA was subsequently analysed 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 2B shows the peak distribution along the complete X-chromosome and in two regions surrounding the ACTB and EIF4A2 positive control genes, respectively (figure 2C and D).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 H3K27ac (Cat. No. 25239) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:4,000.Dot blot analysis using the antibody directed against H3K27ac
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K27ac (Cat. No. 25239) with peptides containing other histone modifications and the unmodified H3K27 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:25,000. Figure 4 shows a high specificity of the antibody for the modification of interest.Western blot analysis using the antibody directed against H3K27ac
Western blot was performed on whole cell (40 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 H3K27ac (Cat. No. 25239). 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 H3K27ac
HeLa cells were stained with the antibody against H3K27ac (Cat. No. 25239) and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K27ac 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.
Concentration: 50 µg/42 µl
Description: Polyclonal antibody raised in rabbit against histone H3, acetylated at lysine 27 (H3K27ac), using a KLH-conjugated synthetic peptide.
Format: Aqueous buffer solution
Formulation: PBS containing 0.05% azide and 0.05% ProClin 300
Immunogen: synthetic peptide
Purification: Affinity purified
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.
Uniprot: P68431
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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