重组Anti-Histone H3 (acetyl K14)抗体[EP964Y] - ChIP Grade (ab52946)

Chromatin was prepared from HeLa cells (treated with 50 ng/ml nocodazole for 14 hours) according to the Abcam X-ChIP protocol. Cells were fixed with EGS (1.5 mM) for 30 minutes then formaldehyde (1%) for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab52946 (red), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (TaqMan approach).

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

Immunocytochemistry/Immunofluorescence analysis of untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and TSA (Trichostatin A) (500ng/ml, 4h) treated HeLa cells labeling Histone H3 (acetyl K14) with purified ab52946 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100, counterstained with ab150120 AlexaFluor^®594 Goat anti-Mouse secondary 1:1000  (2ug/ml). An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Negative Control 1: Rabbit primary antibody and anti-mouse secondary antibody(ab150120)

Negative Control 2: Mouse primary antibody(ab7291) and anti-rabbit secondary antibody(ab150077)

ab52946 at 0.01 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 endometrium carcinoma tissue labeling Histone H3 with ab52946, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on human endometrium carcinoma. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Histone H3 with ab52946, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on rat liver. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Histone H3 with ab52946, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on mouse kidney. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

ab52946 at 1/100 dilution staining Histone H3 (acetyl K14) in human uterus adenocarcinoma tissue by Immunohistochemistry, Paraffin embedded tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.