Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  ChIP - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

Chromatin was prepared from U2OS 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, 2 µg of  ab8898 (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.

  Western blot - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

All lanes : Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - unmodified (ab7228) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K4 (ab7768) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K9 (ab1772) at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K27 (ab1781) at 0.5 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K27 (ab1782) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 15.4 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


Lane 8 shows that Rabbit polyclonal to Histone H3 (tri methyl K9) is blocked by the addition of the immunizing peptide (ab1773). Cross-reactivity with Histone H3 peptide - tri methyl K27 (ab1782) is also shown in Lane 11.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

ab8898 staining human uterine tumour tissue sections by IHC-P.  Sections were fixed in formaldehyde and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking with 5% serum for 30 minutes at 22°C.  The primary antibody was diluted 1/400 and incubated with the sample for 30 minutes at 22°C.  A HRP-conjugated goat anti-rabbit antibody diluted 1/400, was used as the secondary.

This image is courtesy of an Abreview submitted by Dr Patrick Pollard

See Abreview

  ChIP - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

X-Chip assay was performed using nuclear lysates prepared from mouse ES cells. Crosslinking was done for 15 minutes in 1% formaldehyde. Primary antibody was incubated first with peptides ab7228, ab1342, ab1782, ab1773, ab1772 and ab1771 in a chip competition assay and then used in chip at 0.0133µg/ µg chromatin (chip sonication buffer) and incubated with sample for 24 hours at 4°C.

Positive control: ChIP coupled with a peptide competition assay to validate the specificity of the antibody.

Negative control: Genomic region (chr10:79154149-79155200) with no evidence of H3K9me3. 

RT-PCR detection method was used.

Polrmt: PCR primers situated in the coding regions of Polymerase (RNA) mitochondrial (DNA directed).

Agrn: PCR primers situated in the coding regions of Agrin

The image is a courtesy of an anonymous abreview.

  Immunocytochemistry/ Immunofluorescence-Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade(ab8898)

ICC/IF image of ab8898 stained HeLa 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 (ab8898, 0.1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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.

  Immunofluorescence - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

Indian muntjac fibroblast cells stained with anti-Histone H3 tri methyl K9, ab8898, (green, left panel, deconvolution image; red, right panel, epifluorescence image).

The centromeres are enriched in Histone H3 tri methyl K9. There are also additional bands that occur throughout the chromosomes. Note that these images are taken in situ and are imaged under conditions where distinct cytogenetic-like banding patterns have not previously been possible to visualize (e.g., several acetylated antibodies have been reported to be associated with chromosome bands but, although not homogenously distributed along in situ chromosomes, they do not generate distinct banding patterns).

Kirk McManus in the lab of Michael Hendzel, Univeristy of Alberta

  Immunofluorescence - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

A 3-D reconstruction of a mouse embryonic fibroblast cell in metaphase stained with anti-Histone H3 tri methyl K9(green, ab8898) and DAPI (red).

Kirk McManus in the lab of Michael Hendzel, Univeristy of Alberta

  Immunofluorescence - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

These images were kindly submitted by Prof Bryan Turner, University of Birmingham. Undifferentiated Mouse Embryonic Stem cells or cells differentiated for 7 days were incubated with ab8898. The staining is specific for centromeric heterochromatin on metaphase chromosomes.

  Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

ICC/IF image of ab8898 stained HeLa 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 (ab8898, 0.1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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 100% methanol fixed (5 min) HepG2 and MCF7 cells at 0.1µg/ml and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 0.1ug/ml.