Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050)

ChIP analysis of H3K36 trimethylation (me3) along ctt1 in exo2Δ cells.

Strains YAM2400 (WT) and YAM2402 (exo2Δ) were grown to mid-log phase in rich medium and collected before or after addition of 1 mM H₂O₂ for 15 minutes ctt1 mRNA and U3B snoRNA were detected from total RNA using ³²P-labelled oligonucleotides. Data analysis was performed for each position, data were first normalized on act1, then on the level of histone H3, immunoprecipitated from the same chromatin. 

Average values and SEM were calculated from three biological replicates.

*p<0.05; ns, not significant upon t-test.

RNA Pol II phosphoepitopes and histone H3 modifications associated with transcription elongation are altered by sig-7(RNAi).

Equal amounts of lysates from L4440 control RNAi and sig-7(RNAi) embryos were analyzed by western blots probed with ab9050 and antibodies against H3K79me2, and the 5’ end of active genes, H3K4me3. Antibodies against total histone H3 (H3) show similar protein loading.

Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

Cells were fixed with formaldehyde for 10 minutes. The  ChIP was performed with 25 µg of chromatin, 2 µg of ab9050 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH (active) and MYO-D (inactive) promoters and over the ý-Actin gene (active).

Schematic diagram of the ý-Actin gene is shown on the top of the figure.

Black boxes represent exons and thin lines represent introns.

PCR products are depicted as bars under the gene.

ChIP using ab9050 at the Pho4 locus in S. pombe.

Chromatin extract from S.pombe cells was incubated with 5 µg of ab9050 overnight, and then incubated with Protein A beads for 1 hour. The immunoprecipitated DNA was quantified at the Pho4 locus by RT-PCR. No antibody was added to the beads control (red bars). A schematic diagram of the pho4 gene is shown below the graph. The grey box represents the open reading frame and PCR products are depicted by horizontal arrows.

Please see anonymous abreview submitted on 29 October 2008 for additional details.

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ab9050 stained in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

Cells were fixed with 100% methanol for 5 minutes at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9050 at 0.1 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody (colored green) used at 1 µg/ml for 1 hour at room temperature.

DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

ab9050 staining Histone H3 (tri-methyl K36) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed with 100% methanol for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab9050 at 0.1 µg/ml and ab7291 (anti beta-Tubulin) at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat anti-rabbit AlexaFluor^®488 secondary (ab150081) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor^®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

ab9050 staining MAFA in mouse tissue sections (Liver, P5) by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with paraformaldehyde and blocked with 5% BSA for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100) for 3 hours at 25°C. An Alexa Fluor^®555  conjugated Donkey anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

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ab9050 staining rat liver tissue sections by IHC-P.

The section was fixed in formaldehyde and subjected to heat mediated antigen retrieval in pH 6.0 citrate buffer prior to being blocked with 5% serum for 30 minutes at 45°C.  The primary antibody was diluted 1/500 and incubated for 45 minutes at 20°C.  An HRP conjugated goat anti-rabbit was used as the secondary

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ab9050 staining Histone H3 (tri methyl K36) in Saos-2 (Human osteosarcoma cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton in PBS and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/1000) for 1 hour. An Alexa Fluor^®488-conjugated Goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

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