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Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K27) conjugated to keyhole limpet haemocyanin (Sulfosuccinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC)). The exact sequence is proprietary. Clones were positively screened by ELISA against the immunising peptide. Clones were negatively screened against both the unmodified peptide and also against tri methyl K9 Histone H3 peptide.
(Peptide available as
This antibody clone [mAbcam 6002] is manufactured by Abcam.
We have the following conjugates available:
Anti-Histone H3 (tri methyl K27) antibody (Alexa Fluor® 488) [mAbcam 6002] (ab205728)
Anti-Histone H3 (tri methyl K27) antibody (Alexa Fluor® 647) [mAbcam 6002] (ab205729)
Our Abpromise guarantee covers the use of ab6002 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP/Chip||Use at an assay dependent concentration.|
|ChIP||Use 5-10 µg for 25 µg of chromatin.|
|IHC-P||Use at an assay dependent concentration.|
|Flow Cyt||Use at an assay dependent concentration.
ab18392 - Mouse monoclonal IgG3, is suitable for use as an isotype control with this antibody.
|ELISA||Use a concentration of 0.025 - 1 µg/ml.|
|WB||Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K27) peptide (ab1782).
Previously we suggested blocking with BSA as the signal is very weak with milk blocking.
However, in our hands, current batches of ab6002 perform better in western blot using 3% milk block for 1 hour (please see second Western blot image). We recommend optimizing the blocking step in your experiment.
|ICC||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 5 µg/ml.|
Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25µg of chromatin, 5µg of ab6002 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab6002 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406.
ICC/IF image of ab6002 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6002, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
Figure showing the nuclear distribution of H3 (tri-methyl K27) antibody, ab6002 in a) a 46 chromosome, XX cell line, and b) a 49 chromosome, XXXXX cell line.
The location of facultative heterochromatin at the inactive X chromosome is indicated by white arrow heads.
ICC/IF image of ab6002 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6002, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, goat anti-mouse DyLight® 488 (IgG H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Immunofluorescent imaging of human cells (U2OS) with ab6002 reveals broadly dispersed interphase nuclear staining corresponding to trimethylation of K27, with multiple foci of brighter staining, exactly agreeing with published studies of K27-trimethyl IF [See figure 3 of Peters et al Mol Cell 12(6):1577-1589 (2003)] . The lack of nucleolar or cytoplasmic staining background confirms the high specificity of the antibody in this application.
IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes. All blocking and incubation steps carried out at 37 degrees C.
Chromatin was prepared from nuclear lysate of the mouse embryonic stem cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in 1% formaldehyde. The primary antibody was diluted to 0.0133µg/µg chromatin and incubated in ChIP Sonication Buffer with the sample for 24 hours at 4°C. The immunoprecipitated DNA was quantified by real time PCR.
Cdx2: PCR primers situated in the promoter regions of Caudal type homeobox transcription factor 2
Nef3: PCR primers situated in the promoter regions of neurofilament 3
Paraformaldehyde-fixed, paraffin-embedded human invasive breast carcinoma tissue stained for Histone H3 (tri methyl K27) using ab6002 at 1/200 dilution in immunohistochemical analysis.
All batches of ab6002 are tested in ELISA against peptides to different Histone H3 modifications. Results show strong binding to Histone H3 - tri methyl K27 immunising peptide (ab1782), indicating that this antibody specifically recognises the Histone H3 - tri methyl K27 modification. Weak binding is also detected against the Histone H3 - di methyl K27 modification (<12%) (ab1781).
ab17163 - Histone H3 - unmodified
ab1340 - Histone H3 - mono methyl K4
ab7768 - Histone H3 - di methyl K4
ab1342 - Histone H3 - tri methyl K4
ab1771 - Histone H3 - mono methyl K9
ab1772 - Histone H3 - di methyl K9
ab1773 - Histone H3 - tri methyl K9
ab1780 - Histone H3 - mono methyl K27
ab1781 - Histone H3 - di methyl K27
ab1782 - Histone H3 - tri methyl K27