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Polyclonal antibody raised in rabbit against the region of histone H2B containing the acetylated lysine 12 (H2BK12ac), using a KLH-conjugated synthetic peptide.
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
* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 μg per IP.
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).
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.
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.
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.
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.
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.
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Diagenode strongly recommends using this: H2BK12ac polyclonal antibody - Classic (sample size) (Diagenode Cat# C15410212-10 Lot# A2260P). Click here to copy to clipboard.
Using our products in your publication? Let us know!
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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