H2BK12ac polyclonal antibody - Classic (sample size)

Lot	A2260P
Concentration	0.82 µg/µl
Species reactivity	Human
Type	Polyclonal

Purity	Affinity purified
Host	Rabbit
Precautions	This product is for research use only. Not for use in diagnostic or therapeutic procedures.

Applications	Suggested dilution	References
ChIP/ChIP-seq ^*	0.5 - 1 μg per ChIP	Fig 1, 2
ELISA	1:1,000	Fig 3
Dot Blotting	1:5,000	Fig 4
Western Blotting	1:1,000	Fig 5
Immunofluorescence	1:500	Fig 6

H2BK12ac Antibody ChIP Grade

Figure 1. ChIP results obtained with the Diagenode antibody directed against H2BK12ac
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H2BK12ac (Cat. No. C15410212) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010051), using sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 0.5, 1, 2 and, 5 μg per ChIP experiment was analysed. IgG (1 μg/IP) was used as negative IP control. QPCR was performed with primers for a region approximately 1 kb upstream of the GAPDH and ACTB promoters, used as positive controls, and for the coding region of the inactive MYOD1 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).

H2BK12ac Antibody ChIP-seq Grade

H2BK12ac Antibody for ChIP-seq

H2BK12ac Antibody for ChIP-seq assay

H2BK12ac Antibody validated in ChIP-seq

Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H2BK12ac
ChIP was performed on sheared chromatin from 1.5 million HeLaS3 cells using 0.5 μg of the Diagenode antibody against H2BK12ac (Cat. No. C15410212) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the enrichment along the complete sequence and a 1 Mb region of the X-chromosome (fig 2A and B) and in genomic regions of chromosome 7, surrounding the ACTB gene, and of chromosome 12, surrounding the GAPDH gene (fig 2C and D). The position of the amplicon used for ChIP-qPCR is indicated by an arrow.

H2BK12ac Antibody ELISA validation

Figure 3. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H2BK12ac (Cat. No. C15410212) 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 1:38,200.

H2BK12ac Antibody validated in Dot Blot

Figure 4. Cross reactivity tests using the Diagenode antibody directed against H2BK12ac
To test the cross reactivity of the Diagenode antibody against H2BK12ac (Cat. No. C15410212), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H2B. 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.

H2BK12ac Antibody validated in Western Blot

Figure 5. Western blot analysis using the Diagenode antibody directed against H2BK12ac
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 Diagenode antibody against H2BK12ac (Cat. No. C15410212). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.

H2BK12ac Antibody validated in Immunoflurescence

Figure 6. Immunofluorescence using the Diagenode antibody directed against H2BK12ac
HeLa cells were stained with the Diagenode antibody against H2BK12ac (cat. C15410212) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H2BK12ac 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.

Target Description

Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Acetylation of histone H2B is associated with active genes.

実験手法

ELISA
Enzyme-linked immunosorbent assay. Read more

DB
Dot blotting Read more

WB
Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more

ChIP-seq (ab)
Read more

ChIP-qPCR (ab)
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IF
Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more

資料

Datasheet H2BK12ac C15410212 DATASHEET Datasheet description	Download
Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...	Download
Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...	Download

出版物

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Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation
Kieffer-Kwon K.R. et al.
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. Fi...

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H2BK12ac polyclonal antibody - Classic (sample size)

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H2BK12ac polyclonal antibody - Classic (sample size)

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H2BK12ac polyclonal antibody - Classic

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