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

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of ab9050 (blue), and 20µl 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050)

Staining of human tonsil using ab9050 is shown. Antigen retrieval was performed using Tris EDTA at pH9. Nuclei of lymphoid cells in the interfollicular area of a human tonsil stained strongly positive as well as the endothelial cells of blood vessels.

Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050)

ICC/IF image of ab9050 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab9050, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050)

ab9050 staining rat liver tissue sections by IHC-P.  The section was formaldehyde fixed 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.  A HRP conjugated goat anti-rabbit was used as the secondary

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050)

ab9050 staining mouse kidney tissue sections by IHC-P.  The section was formaldehyde fixed and subjected to heat mediated antigen retrieval in pH 6.0 citrate buffer prior to being blocked with 5% serum for 30 minutes at 20°C.  The primary antibody was diluted 1/500 and incubated for 45 minutes at 20°C.  A HRP conjugated goat anti-rabbit was used as the secondary

This image is courtesy of an anonymous Abreview

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

ChIP using ab9050 at the Pho4 locus in S. pombe. Chromatin extract from S.pombe cells was incubated with 5 ug 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.

This image is courtesy of an anonymous Abreview

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

All lanes : Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050) 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 K36 at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K36 (ab1783) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K36 (ab1784) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K36 (ab1785) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K37 (ab24417) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

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

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

ab9050 staining Histone H3 (tri methyl K36) in Human Saos-2 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.

This image is courtesy of an anonymous Abreview