Anti-Histone H3 antibody - Nuclear Marker and ChIP Grade (ab1791)

Histone H3 - ChIP Grade was immunoprecipitated using 0.5mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5 µg of Rabbit polyclonal to and 50 µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.

Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70°C; 10 µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1791.

Secondary Antibody: Mouse anti-rabbit HRP light chain (HRP) (ab99697).

Band: 15kDa; Histone H3 - ChIP Grade

Chromatin from Xenopus laevis oocytes was prepared according to the Abcam X-ChIP protocol.

Oocytes were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 mg of chromatin, 3 mg of ab7834 (anti-H3, light blue) and 3 µg of ab1791 (anti-H3, dark blue), and 20 ml of Protein A/G sepharose beads. A non-specific antibody was used as a control (yellow).

The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).

ab1796 staining Histone H3 in mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with paraformaldehyde, permeabilized with 0.05% Triton X-100 in PBS for 30 minutes and blocked with 5% BSA for 1 hour; antigen retrieval was by heat mediation in sodium citrate pH 6. Samples were incubated with the primary antibody (1/500 in blocking buffer) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG polyclonal (1/400) was used as the secondary antibody.

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Histone H3 immunogold detection in HeLa cells.
Ulltra-thin sections of paraformaldehyde-fixed, Lowicryl K4M embedded cells were incubated sequentially with ab1791 antibody and an anti-rabbit antibody coupled to 10 nm gold particles. Notice absence of chromatin within Interchromatin Granules (IG), a reservoir of spliceosomal snRNPs, and high labeling of patches of condensed chromatin close to the nuclear envelope (arrows). Nu: nucleus, Cyt: cytoplasm.

Image courtesy of Gerard Pierron, IGR-Villejuif, France.

Sample : human
Type : HeLa cells
Fixative : either paraformaldehyde 4% or 1.6% glutaraldehyde in 0.1M Millonig’s buffer.
Embedding : in Lowicryl 4KM at -20°C under UV.
Ultrathin-sections deposited on formvar-coated carbonated EM-grids.
Blocking step: 5% BSA for 30 seconds at RT.
Primary antibody: ab1791 diluted 1/50 in PBS, for 1h at RT.
Secondary antibody: BBI International anti-rabbit

Blocked with 3% milk for 1 hour at 25°C.

Incubated with the primary antibody for 16 hours at 4°C in 3% milk in TBS-tween.

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Rabbit polyclonal to Histone H3 (ab1791) at 1/5000 on S. cerevisiae whole cell lysate (40 ug per lane).

Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000.  Blots plus primary antibodies were either incubated overnight at 4°C or at RT for 2 hours. Blots were washed 6X for 10 minutes each in PBS with 0.1% Tween-20 before addition of secondary antibodies. Secondary antibodies were diluted 1/2,000 in blocking buffer and incubated with blots for 2 hours at RT. Secondary blots were washed 4X for 10 minutes each in PBS with 0.1% Tween-20 and 2X for 10 minutes each in PBS.

Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 ab1791 (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.

The ChIP was performed with chromatin from mouse gut cell lysate and ab1791 at 1/250 dilution.

Negative control: No antibody was used (right bar).

The immunoprecipitated DNA was quantified by real time PCR.

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Paraffin-embedded rat brain tissue stained for Histone H3 using ab1791 at 1/8000 dilution in immunohistochemical analysis.

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ab1791 staining Histone H3 in human infantile fibromatosis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with formaldehyde and blocked with 1% FBS/BSA for 3 hours at room temperature; antigen retrieval was by heat mediation in Tris pH 9. Samples were incubated with primary antibody (1/100 in TBS + 1% BSA + 1% FBS) for 16 hours. An un-diluted HRP-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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ab1791 staining Histone H3 (red) in rat brain tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with formaldehyde, permeabilized with 0.1% TBS-TritonX and blocked with 10% serum for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with the primary antibody (1/500 in 10% normal goat serum) for 24 hours at 24°C. An Alexa Fluor^® 594-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

Green - Nucleus staining.
Red - Histone H3 staining.