Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 4 µg of ab176799 (EPR16596). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. 

 

Additional screenshots of mapped reads can be downloaded here. 

Chromatin was prepared from HeLa (Human epithelial cells from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab176799 (red), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

Dot blot analysis of Histone H3 (acetyl K4) labeled with ab176799 at 1/1000 dilution.

Lane 1: H3 (acetyl K4) peptide

Lane 2: H3 (formylK4) peptide

Lane 3: H3 (methylK4) peptide

Lane 4: H3 (crotonylK4) peptide

Lane 5: Unmodified H3 peptide

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated was used as secondary antobody at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

All lanes : Anti-Histone H3 (acetyl K4) antibody [EPR16596] - ChIP Grade (ab176799) at 1/10000 dilution

Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) treated with 500 ng/mlTrichostatin A for 4 hours whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Histone H3 (acetyl K4) antibody [EPR16596] - ChIP Grade (ab176799) at 1/10000 dilution

Lane 1 : Untreated NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embyro fibroblast cells) treated with 500 ng/ml Trichostatin A for 4 hours whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells, treated with Trichostatin A (500 ng/ml) for 4 hours or untreated, labeling Histone H3 (acetyl K4) with ab176799 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line. The expression increased after treatment with Trichostatin A (500 ng/ml) for 4 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab176799 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.