Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade

10 product images

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  ChIP - Anti-Histone H3 (acetyl K14) antibody [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).

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  Western blot - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

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

  All lanes: Western blot - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946) at 1/1000 dilution

  Lane 1: C6 (Rat glial tumor glial cell) treated with Trichostatin A whole cell lysates at 15 µg

  Lane 2: 293T (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

  Lane 3: HEL (Human Erythroleukemia erythroblast) whole cell lysates at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

  Exposure time: 60s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  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 Anti-DLL3 antibody [EPR22592-18] 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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  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 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). Nuclei counterstained with DAPI (blue).
  

  Negative Control 1: Rabbit primary antibody and anti-mouse secondary antibody(Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120)

  Negative Control 2: Mouse primary antibody(Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) and anti-rabbit secondary antibody(Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077)

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  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 Anti-DLL3 antibody [EPR22592-18] 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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  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 Anti-DLL3 antibody [EPR22592-18] 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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).

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  Western blot - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  Different batches of ab52946 were tested on C6 (Rat glial tumor glial cell) treated with Trichostatin A lysate at 1.2 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 15 kDa.

  All lanes: Western blot - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  Predicted band size: 15 kDa

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  Western blot - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  All lanes: Western blot - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946) at 1/2000 dilution

  Lane 1: C6 (Rat glial tumor cell line) cell lysates untreated at 10 µg

  Lane 2: C6 cell lysates treated with Trichostatin A at 10 µg

  Secondary

  All lanes: goat anti-rabbit HRP at 1/2000 dilution

  Predicted band size: 15 kDa

  Observed band size: 11 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  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.

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  Peptide Array - Anti-Histone H3 (acetyl K14) antibody [EP964Y] - ChIP Grade (ab52946)

  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