All lanes : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 peptide (ab17163) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

Lysates/proteins at 0.25 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa

why is the actual band size different from the predicted?


Exposure time: 3 minutes

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.

In mice, the X-inactivation process is reversed naturally by X-reactivation in blastocysts and germ cells and in culture in pluripotent stem cells. The image shows late blastocyst-stage mouse embryos consisting of three cell types: epiblast (NANOG-positive, cyan), primitive endoderm (GATA4-positive, red) and trophectoderm (CDX2-positive, green). The inactive X-chromosome (H3K27me3-positive, yellow dots) is reactivated only in the epiblast (cells without yellow spots), which will form the embryo. The germ cell factor PRDM14 and the long noncoding RNA Tsix collaborate during the X-reactivation process in blastocysts and pluripotent stem cells and thereby link epigenetic with cellular reprogramming events.

Image is courtesy of Bernhard Payer, runner-up of the immunofluorescence imaging competition 2017.

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 peptide (ab1782), indicating that this antibody specifically recognizes the Histone H3 tri methyl K27 modification. Weak binding is also detected against the Histone H3 di methyl K27 modification (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

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 5 µg of ab6002 (blue), and 20 µl 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.
    

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.

Lanes 1-3 : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml (2% BSA)
Lanes 4-6 : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml (3% MILK)

Lanes 1 & 4 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lanes 2 & 5 : EED-/- mouse ES Whole Cell Lysate
Lanes 3 & 6 : WT mouse ES Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 12 minutes

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 2% Bovine Serum Albumin (lanes 1-3) and 3% milk (lanes 4-6) 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.

Chromatin was prepared from K562 cells. Cells were fixed with formaldehyde for 10 min. A GATA1 antibody was used as the positive control and a Rabbit IgG was used as the negative control. Incubation with primary antibody was in Immuno Precipitation Dilution buffer for 16 hours at 4°C. The immunoprecipitated DNA was quantified by real time PCR. 

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