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Applications: ChIP (15 µl/ChIP)
ELISA (1:1000 - 1:4000)
DB (1:20,000)
WB (1:250)
IF (1:500)
Assay Conditions: ChIP results obtained with the antibody directed against H3K9acS10p
ChIP assays were performed using human osteosarcoma (U2OS) cells, the antibody against H3K9acS10p (Cat. # 25267) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 5 and 15 µl per ChIP experiment was analyzed. IgG (5 µg/IP) was used as negative IP control. QPCR was performed with primers for the ALDOA (fructose-bisphosphate aldolase A) promoter and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).Determination of the titer
To determine the titer, an ELISA was performed using a serial dilution of the antibody against human H3K9acS10p (Cat. # 25267). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:89,000.Cross reactivity test using the antibody directed against H3K9acS10p
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K9acS10p (Cat. # 25267) with peptides containing other modifications of histone H4 and H3 or unmodified histone H3 sequences. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the double modification.Western blot analysis using the antibody directed against H3K9acS10p
Histone extracts of HeLa cells (15 µg) were analyzed by Western blot using the antibody against H3K9acS10p (Cat. # 25267) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right.Immunofluorescence using the antibody directed against H3K9acS10p
HeLa cells were stained with the antibody against H3K9acS10p (Cat. # 25267) and with DAPI. Cells were fixed with 4% formaldehyde for 10 min. and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K9acS10p 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.
Background: Acetylation of K9 and phosphorylation of S10 of histone H3 are associated with active gene transcription.
Concentration: 100 µl
Cross Reactivity: Specific for H3K9acS10p. Does not cross-react with H3K9ac, H3K9me(1, 2, or 3), H3S10p, or H3K9ac14ac.
Description: Polyclonal antibody raised in rabbit against histone H3 containing the acetylated lysine 9 and the phosphorylated serine 10 (H3K9acS10p), using a KLH-conjugated synthetic peptide.
Format: Aqueous solution
Formulation: Whole antiserum from rabbit containing 0.05% azide
Immunogen: synthetic peptide
Purification: Whole serum
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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