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Synthetic peptide derived from residues 1 - 100 of Human Histone H3, tri methylated at K27.
. Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide. Clones were negatively screened against both the corresponding unmodified peptide and also against a peptide corresponding to tri methylated K9 of Histone H3.
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.
|IP||Use at an assay dependent concentration.|
|ChIP/Chip||Use at an assay dependent dilution.|
|ChIP||Use 5-10 µg for 25 µg of chromatin.|
|IHC-P||Use at an assay dependent dilution.|
|Flow Cyt||Use at an assay dependent dilution.|
|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). Block with BSA. The signal is very weak with milk blocking.|
|ICC||Use at an assay dependent dilution.|
|ICC/IF||Use a concentration of 5 µg/ml.|
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.
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.
ELISA using ab6002 at antibody concentrations between 1/500 and 1/8000.
The red line indicates binding to the tri methyl K27 peptide (ab1782). Binding to the following peptides was not seen:
Unmodified K27 (ab2623),
mono methyl K27 (ab1780),
di methyl K27 (ab1781),
mono methyl K9 (ab1771),
di methyl K9 (ab1772),
tri methyl K9 (ab1773),
mono methyl K4 (ab1340),
di methyl K4 (ab7768),
tri methyl K4 (ab1342).
This indicates the specificity of ab6002 for tri methyl K27 of Histone H3.
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
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.
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