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Tri-Methyl-Histone H3 (Lys9) Polyclonal Antibody
Antibody target was verified by Relative expression to ensure the antibody binds to the antigen stated.
Catalog # 49-1008
(USD) 349.00, 50 µg
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Immunofluorescence analysis of Tri-Methyl-Histone H3 (Lys9) was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Rabbit Polyclonal Antibody (Product # 49-1008) at 5ug/ml in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Immunofluorescence study using the anti-H3K9me3 polyclonal antibody. NIH3T3 cells (mouse fibroblasts) were formaldehyde fixed, permeabilized with Triton® X-100 (Dow Chemical Co) and blocked with PBS containing 2.5% BSA. (A) Cells were labeled with the anti-H3K9me3 antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by FITC-labeled goat anti-rabbit secondary antibody. (B) Nuclei were stained using the DNA-specific stain DAPI. Both the anti-H3K9me3 antibody and DAPI produced clear nuclear staining. The dense signals obtained with both probes characterize the distribution pattern of H3K9me3, which is linked to the transcriptionally inactive, condensed pericentric heterochromatin.
Cross reactivity tests using the anti-H3K9me3 antibody. A dot blot analysis was performed to test the cross reactivity of the anti-H3K9me3 polyclonal antibody (Product # 49-1008) with other histones and other histone modifications. Other histone modifications include mono- and dimethylation of the same lysine and mono-, di-, and trimethylation of lysine 27, 36, and 79. 100 to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane and detected with the antibody diluted 1:20,000. This figure shows the high specificity of the purified antibody sample with the histone modification of interest.
Western blot analysis was performed on acid extracts (20 µg lysate) of HeLa (Lane 1), C2C12 (Lane 2), NIH/3T3 (Lane 3), K-562 (Lane 4) and Jurkat (Lane 5). The blot was probed with Anti-Tri-Methyl-Histone H3 (Lys9) (Product # 49-1008, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 17 kDa band corresponding to Tri-Methyl-Histone H3 (Lys9) was observed across the cell lines tested.
Western blot analysis using the rabbit anti-H3K9me3 polyclonal antibody. Histone extracts of HeLa cells (20 µg) were analyzed by western blot using the anti-H3K9me3 antibody (Product # 49-1008), diluted 1:1,000 in TBS-Tween containing 5% milk. The molecular weight marker (in kDa) is shown on the left; the location of the protein of interest is indicated on the right.
ChIP results obtained with the rabbit anti-H3K9me3 polyclonal antibody. ChIP assays were performed using HeLa cells, the rabbit anti-H3K9me3 polyclonal antibody (Product # 49-1008) and optimized real time PCR primer sets for qPCR. Each ChIP assay used sheared chromatin from 1.5 million cells and 5 µg of H3K9me3 antibody. IgG (5 µg/IP) was used as a negative IP control. Trimethylation of H3K9 is a marker for heterochromatin. Therefore, we used the promoter of a housekeeping gene c-fos, which is actively transcribed, as a negative control target. Sat-2, present in heterochromatin, is used as a positive control target. Recoveries (expressed as percentage of input) are shown. Bars 1 and 3 represent the recovery (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis) using the H3K9me3 antibody of the c-fos promoter and Sat-2, respectively. Bars 2 and 4 represent the recovery using the IgG negative control.
Enrichment of endogenous Tri-Methyl-Histone H3 (Lys9) protein at specific gene loci using Anti-Tri-Methyl-Histone H3 (Lys9) Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-Tri-Methyl-Histone H3 (Lys9) Rabbit Polyclonal Antibody (Product # 49-1008, 3 ug) on sheared chromatin from 2 million HeLa cells using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR with PCR primer pairs for the promoters of SAT2 satellite repeats, SATa used as positive, and C-FOS, ALDOA, GAPDH used as negative target genes/binding sites. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
The specificity of the antibody, Anti-Tri-Methyl-Histone H3 (Lys9) Polyclonal Antibody (Product # 49-1008, 0.25 µg/ml) to Histone H3K9me3 peptide was confirmed using MODified™ Histone Peptide Array (Active Motif, 13001). Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) was used as the secondary antibody. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). The dot blot data obtained (lower panel) was analyzed using Array Analyze software as per the manufacturer's instructions (top panel).
To determine the titer, an ELISA was performed using anti-H3K9me3 crude serum, the anti-H3K9me3 polyclonal antibody (Product # 49-1008), and the column flow through obtained from the antibody purification step. The antigen used to coat the ELISA plate was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be 1:13, 500.
Antibody specificity was demonstrated by detection of enrichment of the target protein at specific gene loci. Chromatin Immunoprecipitation (ChIP) was performed using Anti-Tri-Methyl-Histone H3 (Lys9) Polyclonal Antibody (Product # 49-1008) with relevant positive (SAT2 satellite repeats, SATa) and negative (C-FOS, ALDOA, GAPDH) target genes/ binding sites.
Antibody specificity for modified targets can be established using peptide arrays by quantifying detection of the target protein along with closely related proteins. Peptide array of Histone H3K9me3 using Anti-Tri-Methyl-Histone H3 (Lys9) Polyclonal Antibody: An array of the specific peptide and other relevant peptides when tested using Anti-Tri-Methyl-Histone H3 (Lys9) Polyclonal Antibody (Product # 49-1008), showed that the Histone H3K9me3 modification was specifically recognized by the antibody.
Figure 5 UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means +- SD of three independent experiments. * P<0.05 (t-test). (C) Micrococcal nuclease assay of 5637-NC, 5637-iUCA1. Same amounts of DNA were digested with micrococcal nuclease and electrophoresed. The image shows that nuclease digestion produced a laddering pattern. It is evident that DNA from 5637-iUCA1 is more sensitive to nuclease digestion. (D) Western blotting to detect histone proteins H3K4m3, H3K9m3 in 5637-NC, 5637-iUCA1. (E) ChIP analysis of BRG1 binding to the p21 promoter in EJ-BRG1, EJ-BRG1-UCA1. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means +- SD of three independent experiments. Overexpression of UCA1 in EJ-BRG1 led to decreased occupancy of p21 promoter by BRG1, * P<0.05 (t-test). UCA1, u
Published figure using Tri-Methyl-Histone H3 (Lys9) polyclonal antibody (Product # 49-1008)
ChIP assay (ChIP)
1.5 µg/10^6 cells
1:100 to 1:500
Western Blot (WB)
Peptide Array (Array)
Host / Isotype
rabbits against histone H3, trimethylated at lysine 9 [K9me3], using a KLH-conjugated synthetic peptide.
<0.1% sodium azide
-20° C, Avoid Freeze/Thaw Cycles
Histone H3 is one of the DNA-binding proteins found in the chromatin of all eukaryotic cells. H3 along with four core histone proteins binds to DNA forming the structure of the nucleosome. Histones play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. Post tranlationally, histones are modified in a variety of ways to either directly change the chromatin structure or allow for the binding of specific transcription factors. The N-terminal tail of histone H3 protrudes from the globular nucleosome core and can undergo several different types of post-translational modification that influence cellular processes. These modifications include the covalent attachment of methyl or acetyl groups to lysine and arginine amino acids and the phosphorylation of serine or threonine.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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Protein Aliases: H3 histone family, member A; H3a; H3K9me3; HIST1H3A; Histone 1; histone 1, H3a; Histone H3.1; Histone H3/a; Histone H3/b; Histone H3/c; Histone H3/d; Histone H3/f; Histone H3/h; Histone H3/i; Histone H3/j; Histone H3/k; Histone H3/l
Gene Aliases: H3/A; H3FA; H3FB; H3FC; H3FD; H3FF; H3FH; H3FI; H3FJ; H3FK; H3FL; HIST1H3A; HIST1H3B; HIST1H3C; HIST1H3D; HIST1H3E; HIST1H3F; HIST1H3G; HIST1H3H; HIST1H3I; HIST1H3J
UniProt ID: (Human) P68431
Entrez Gene ID: (Human) 8350, (Mouse) 360198
DNA binding protein
nucleic acid binding
Suggested Secondary Antibodies
Material safety data sheets (MSDS)