View (157) other Histone H3 antibodies
Tri-Methyl-Histone H3 (Lys27) Polyclonal Antibody
Antibody target was verified by Relative expression to ensure the antibody binds to the antigen stated.
Catalog # A15024
(USD) 349.00, 50 µg
Save to list
Product Image Gallery
Immunofluorescence analysis of Tri-Methyl-Histone H3 (Lys27) 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 # A15024) at 1:100 dilution 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 (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.
Mouse NIH3T3 cells were stained with antibody against H3K27me3 (Product # A15024) 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 H3K27me3 antibody (left) diluted 1:200 in blocking solution followed by an antirabbit 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.
Western blot analysis was performed on acid extracts (20 µg lysate) of HeLa (Lane 1), NIH/3T3 (Lane 2), HCT 116 (Lane 3), C2C12 (Lane 4), U-2 OS (Lane 5), Hep G2 (Lane 6) and MCF7 (Lane 7). The blot was probed with Anti-Tri-Methyl-Histone H3 (Lys27) (Product # A15024, 1:500 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 (Lys27) was observed across the cell lines tested.
ChIP assays were performed using human HeLa cells, the antibody against H3K27me3 (Product # A15024) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP” kit (Product # 4487119), using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes EIF4A2 and GAPDH as negative controls, and for the coding regions of the inactive genes MYT1 and TSH2B as positive controls. This figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K27me3 is preferably present at inactive genes.
Enrichment of endogenous Tri-Methyl-Histone H3 (Lys27) protein at specific gene loci using Anti-Tri-Methyl-Histone H3 (Lys27) Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-Tri-Methyl-Histone H3 (Lys27) Rabbit Polyclonal Antibody (Product # A15024, 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 MYOD and SAT Alpha 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.
ELISA was performed to determine the titer of the H3K27me antibody, using a serial dilution directed against H3K27me3 (Product # A15024). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:3, 500.
The specificity of the antibody, Anti-Tri-Methyl-Histone H3 (Lys27) Polyclonal Antibody (Product # A15024, 1:2000 dilution) to Histone H3K27me3 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).
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K27me3 (Product # A15024) with peptides containing other modifications of histone H3 and H4 and the unmodified H3K27 sequence. 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 1:5,000. This figure shows a high specificity of the antibody for the modification of interest. Please note that that antibody also recognizes the modification if S28 is phosphorylated.
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 (Lys27) Polyclonal Antibody (Product # A15024) with relevant positive (MYOD, SAT A) and negative (C-FOS, ALDOA, GAPDH) target genes/ binding sites.
ChIP assay (ChIP)
1.5 µg/10^6 cells
Western Blot (WB)
Peptide Array (Array)
Host / Isotype
rabbit immunized with the region of the histone H3 containing the trimethylated lysine 27 (H3K27me3)
0.05% 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.
Have you cited this antibody in a publication?
so we can reference it in this datasheet.
Protein Aliases: H3 histone family, member A; H3a; H3k27; H3K27me3; 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)