Western blot - Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

All lanes : Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812) 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 (ab2903) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K9 (ab16635) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K14 at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K18 (ab24003) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K27 (ab24404) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K23 (ab48359) 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 1/5000 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 : 30 seconds

ChIP - Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

Chromatin was prepared from Hela 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  ab10812 (blue), and 20µl 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). Primers and probes are located in the first kb of the transcribed region.
    

Immunocytochemistry/ Immunofluorescence - Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

ICC/IF image of ab10812 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 (ab10812, 1µg/ml) overnight at +4ºC. 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) Hek293, HepG2, and MCF-7 cells at 5µg/ml.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

IHC image of Histone H3 (acetyl K9) staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab10812, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Immunocytochemistry - Histone H3 (acetyl K9) antibody (ab10812)

SK-N-SH cells fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and incubated for 1 hour with ab10812 (1/1000). ab10812 clearly stained the nucleus (red). The cells were counterstained with DAPI (blue). 100x magnification.

Darin McDonald, Cross Cancer Institute

Flow Cytometry - Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

ab10812 staining mouse embryonic stem cells by flow cytometry (gated on all living cells). The cells were incuabted with the antibody at 0.25ug/1.5 x 10⁵ cells in a permeabilization buffer. A PE conjugated goat anti-rabbit antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Prof Albrecht Müller

Immunocytochemistry/ Immunofluorescence - Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

ab10812 staining Histone H3 (acetyl K9) in the root tips of Arabidopsis thaliana by Immunocytochemistry/ Immunofluorescence. Cells were fixed in 2% paraformaldehyde for 30 minutes at room temperature. Blocking and permeabilization was carried with 4% BSA solution containing 0,5% Triton X-100 in PBS at room temperature for 45 minutes. Slides were washed in PBS and incubated with primary antibody at a 1/200 dilution in 1% PBS for 1 hour at 37ºC. Slides were washed in PBS and incubated with the secondary antibody ab6639 Goat polyclonal to Rabbit IgG - H&L (Cy3 ®) at a 1/500 dilution in 1% BSA in PBS for 1 hour at 37ºC. Slides were counterstained with DAPI (2µg/mL) for 10 minutes at room temperature.

Left image: DAPI stained interphase nucleus with prominent chromocenters
Middle image: Distribution of Histone H3 (acetyl K9) in the nucleus (arrows indicate absence of signal from chromocenters)
Right image: Merged image

This image was kindly supplied by Ms Vedrana Vicic by Abreview

Western blot - Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812)

All lanes : Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812) at 1/1000 dilution

Lane 1 : Tissue lysate prepared from rat dorsal spinal cord at 5 µg
Lane 2 : Tissue lysate prepared from rat dorsal spinal cord at 5 µg
Lane 3 : Tissue lysate prepared from rat dorsal spinal cord at 8 µg
Lane 4 : Tissue lysate prepared from rat dorsal spinal cord at 8 µg
Lane 5 : Tissue lysate prepared from rat dorsal spinal cord at 8 µg with Histone H3 peptide - acetyl K9 (ab16635)

Secondary
HRP conjugated rabbit polyclonal
developed using the ECL technique

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


Exposure time : 2 minutes

Each sample is from a different extract.

Image courtesy of an anonymous Abreview.