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Monoclonal antibody raised in rabbit against the region of histone H3 containing the acetylated lysine 9 (H3K9ac), using a KLH-conjugated synthetic peptide.
Lot
003
Concentration
1 μg/μl
Species reactivity
Human, wide range expected.
Type
Monoclonal ChIP-seq grade
Purity
Affinity purified polyclonal antibody.
Host
Rabbit
Storage Conditions
Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.
Storage Buffer
PBS containing 50% glycerol, 1% BSA and 0.09% azide.
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 IP
Fig 1, 2
Dot Blotting
1:10,000
Fig 3
Western Blotting
1:1,000
Fig 4
Immunofluorescence
1:200
Fig 5
*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 monoclonal antibody directed against H3K9ac ChIP was performed with the Diagenode antibody against H3K9ac (cat. No. C15210015) on sheared chromatin from 500,000 HeLaS3 cells using the “iDeal ChIP-seq” kit (cat. No. C01010051). A titration of the antibody consisting of 0.5, 1, and 2 µg per ChIP experiment was analysed. IgG (1 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the promoters of the GAPDH and EIF4A2 genes, used as positive controls, and for the MYOD1 gene and the Sat2 satellite repeat, used as negative controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
A.
B.
C.
D.
Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K9ac ChIP was performed on sheared chromatin from 500,000 HeLaS3 cells using 0.5 µg of the Diagenode antibody against H3K9ac (cat. No. C15210015) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. 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 signal distribution along the X chromosome and a zoomin to a 600 kb region (fig 2A and B) and in two genomic regions containing the GAPDH and EIF4A2 genes (fig 2C and D).
Figure 3. Cross reactivity tests using the Diagenode monoclonal antibody directed against H3K9ac To test the cross reactivity of the Diagenode antibody against H3K9ac (cat. No. C15210015), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K9. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:10,000. Figure 2 shows a high specificity of the antibody for the modification of interest.
Figure 4. Western blot analysis using the Diagenode monoclonal antibody directed against H3K9ac Western blot was performed on whole cell extracts (40 µg) from HeLa cells using the Diagenode antibody against H3K9ac (cat. No. C15210015). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.
Figure 5. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K9ac HeLa cells were stained with the Diagenode antibody against H3K9ac (cat. No. C15210015, red) diluted 1:200. Actin was stained with fluorescein phalladoin (green).
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.
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