H3T11 Antibody - ChIP Grade (C15410309)

Lot	A2288P
Concentration	1.4 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 ^*	5 μg/ChIP	Figure 1
ELISA	1:1,000	Figure 2
Dot Blotting	1:20,000	Figure 3
Western Blotting	1:1,000	Figure 4
Immunofluorescence	1:200	Figure 5

H3T11p Antibody ChIP Grade

Figure 1. ChIP results obtained with the Diagenode antibody directed against H3T11p
ChIP assays were performed using human HeLa cells, treated with colcemid, the Diagenode antibody against H3T11p (cat. No. C15410309) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010055), 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 (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the EIF4A2 and GAPDH promoters, used as positive controls, and for the coding region of the inactive MYT1 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).

H3T11p Antibody ELISA validation

Figure 2. 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 H3T11p (cat. No. C15410309) 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 2), the titer of the antibody was estimated to be 1:25,700.

H3T11p Antibody valiadted in Dot Blot

Figure 3. Cross reactivity tests using the Diagenode antibody directed against H3T11p
To test the cross reactivity of the Diagenode antibody against H3T11p (cat. No. C15410309), a Dot Blot analysis was performed with peptides containing other histone phosphorylations and the unmodified H3T11. One hundred to 0.2 pmol of the respective peptides 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 modification of interest.

H3T11p Antibody valiadted in Western Blot

Figure 4. Western blot analysis using the Diagenode antibody directed against H3T11p
Western blot was performed on whole cell extracts from untreated HeLa cells (25 μg, lane 1), on histone extracts from HeLa cells treated with colcemid (15 μg, lane 2), and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3T11p (cat. No. C15410309). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.

H3T11p Antibody valiadted in Immunofluorescence

Figure 5. Immunofluorescence using the Diagenode antibody directed against H3T11p
HeLa cells were treated with colcemid and stained with the Diagenode antibody against H3T11p (cat. No. C15410309) 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 labelled with the H3T11p antibody (left) diluted 1:200 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, H2A, 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.

Applications

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

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

ChIP-qPCR (ab)
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Documents

Datasheet H3T11p C15410309 DATASHEET
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