ChIP - Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191)

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.

Immunofluorescence - Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191)

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).

Danièle Roche, Geneviève Almouzni's lab, UMR218

Western blot

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

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

Lysates/proteins at 0.5 µg/ml per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size : 15.4 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)

Immunocytochemistry/ Immunofluorescence - Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191)

ICC/IF image of ab1191 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 (ab1191, 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 MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.

Western blot - Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191)

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 Histone H3 peptide - acetyl K9 (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 Histone H3 peptide - acetyl K18 (ab24003) at 0.5 µg/ml
Lane 6 : HeLa Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K23 (ab48359) at 0.5 µg/ml
Lane 7 : HeLa Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K27 (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 Histone H4 peptide - acetyl K12 (ab15662) at 0.5 µg/ml

Lysates/proteins at 2.5 µg per lane.

Secondary
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