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 ab1191 (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). Primers and probes are located in the first kb of the transcribed region.

All batches of ab1191 are tested in Peptide Array against peptides to different Histone H3 and H4 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - acetyl K18 peptide (ab24003), indicating that this antibody specifically recognises the Histone H3 - acetyl K18 modification.

ab17163 - Histone H3 - unmodified

ab16635 - Histone H3 - acetyl K9

ab15591 - Histone H3 - acetyl K14

ab24003 - Histone H3 - acetyl K18

ab48359 - Histone H3 - acetyl K23

ab24404 - Histone H3 - acetyl K27

ab41409 - Histone H3 - acetyl K36

ab15662 - Histone H4 - acetyl K12

ab1191 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab1191 at 0.1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

All lanes : Anti-Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191) at 1 µg/ml

Lane 1 : HeLa Histone Preparation Nuclear Lysate
Lane 2 : HeLa Histone Preparation Nuclear Lysate with Histone H3 peptide - unmodified R17 at 0.5 µg/ml
Lane 3 : HeLa Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K9) peptide (ab16635) at 0.5 µg/ml
Lane 4 : HeLa Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K14 at 0.5 µg/ml
Lane 5 : HeLa Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K18) peptide (ab24003) at 0.5 µg/ml
Lane 6 : HeLa Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K23) peptide (ab48359) at 0.5 µg/ml
Lane 7 : HeLa Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K27) peptide (ab24404) at 0.5 µg/ml
Lane 8 : HeLa Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K36 at 0.5 µg/ml
Lane 9 : HeLa Histone Preparation Nuclear Lysate with Human Histone H4 (acetyl K12) peptide (ab15662) at 0.5 µg/ml

Lysates/proteins at 2.5 µg per lane.

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

Developed using the ECL technique.

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?


Exposure time: 1 minute

L929, 3T3 and HeLa cells fixed with 2% paraformaldehyde in PBS were incubated for 5 min in 0.2% Triton X-100 in PBS before a blocking step in 5% BSA, 0.1% Tween 20 in PBS (blocking buffer). Ac K18-H3 (ab1191) antibody was added diluted at 1/500 in blocking buffer for a 45 min incubation at room temperature. For visualization, fluorescein isothiocyanate (FITC) anti rabbit secondary antibody was used diluted at 1/800. DAPI (Sigma) in 0.1% Tween 20 in PBS was used in the final washing steps to stain DNA. Vectashield (Vector Laboratories Inc.) was used as mounting medium.
The ac K18-H3 antibody revealed a granular staining throughout the nucleoplasm, excluding regions that show an intense DAPI staining (mostly pericentric heterochromatin in mice cells).

IHC image of Histone H3 (acetyl K18) staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab1191, 1µg/ml, for 15 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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191) at 1 µg/ml

Lane 1 : Histone prep
Lane 2 : Histone prep with Human Histone H3 (acetyl K18) peptide (ab24003) at 1 µg/ml

Lysates/proteins at 0.5 µg/ml per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size: 15.4 kDa