重组Anti-Histone H3 (acetyl K18)抗体[EPR16595] - ChIP Grade (ab177870)

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 ab177870 [EPR16595]. 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 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab177870 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of Rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers and probes are located in the first kb of the transcribed region.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (acetyl K18) with ab177870 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing nuclear staining on HeLa cell line. Acetylation levels 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-alpha Tubulin antibody [DM1A] - Loading Control) at 1/500 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/500 dilution (red).
The negative controls are as follows;
-ve control 1: ab177870 at 1/2000 dilution followed by ab150120 at 1/500 dilution.
-ve control 2: ab7291 at 1/500 dilution followed by ab150077 at 1/400 dilution.

ab177870 at 0.02 μg/mL was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate). A Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated at 1/50000 dilution was used as the secondary antibody. Washing buffer and time was protein Array Washing Buffer, 2*10mins and 2*5mins and blocking buffer was 5% BSA in TBST.
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

Scanner details: Innoscan 710

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3 (acetyl K18) with ab177870 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on glandular epithelium of colon tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Dot blot analysis using ab177870 at 1/1000 dilution and Goat Anti-Rabbit IgG H&L (HRP) (ab97051) as secondary antibody at 1/100,000 dilution.

Lane 1: Histone H3(acetyl K4 + K9 + K14 + K18) peptide

Lane 2: Histone H3(acetyl K4) peptide

Lane 3: Histone H3(acetyl K9) peptide

Lane 4: Histone H3(acetyl K14) peptide

Lane 5: Histone H3(acetyl K18) peptide

Lane 6: Histone H3 unmodified peptide

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Histone H3 (acetyl K18) with ab177870 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on glandular epithelium of mouse colon tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling Histone H3 (acetyl K18) with ab177870 at 1/500 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on Rat cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is prediluted Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.