Anti-HDAC1 antibody - Nuclear Loading Control

11 product images

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  Immunoprecipitation - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to HDAC1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
  The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
  Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19845.
  Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).
  Band: 60ka: HDAC1.

  All lanes: Immunoprecipitation - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  Predicted band size: 55 kDa

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  Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Western blot staining using rabbit Anti-HDAC1 antibody

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: HDAC1 knockout HAP1 cell lysate (20 μg)
  Lane 3: HeLa cell lysate (20 μg)
  Lane 4: Human breast carcinoma lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab19845 observed at 65 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  ab19845 was shown to recognize HDAC1 when HDAC1 knockout samples were used, along with additional cross-reactive bands. Wild-type and HDAC1 knockout samples were subjected to SDS-PAGE. ab19845 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  Predicted band size: 55 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-HDAC1 antibody

  ab19845 staining HDAC1 in wild-type HAP1 cells (top panel) and HDAC1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab19845 at 0.5μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
  

  Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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  Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Western blot staining using rabbit Anti-HDAC1 antibody

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: HDAC1 knockout HAP1 cell lysate (20 μg)
  Lane 3: HeLa cell lysate (20 μg)
  Lane 4: Human breast carcinoma lysate (20 μg) or K562 lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab19845 observed at 65 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  This western blot image is a comparison between ab19845 and a competitor's top cited rabbit polyclonal antibody.

  All lanes: Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  Predicted band size: 55 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-HDAC1 antibody

  ICC/IF image of ab19845 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab19845, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
  

  Panel A shows localisation of ab19845 to the nuclei, Panel B has the Alexa Fluor® 488 channel removed for comparison.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-HDAC1 antibody

  The image shows staining of human tonsil tissue using ab19845. Staining was nuclear and was equally successful using Tris EDTA pH9 or Citrate pH6 for antigen retrieval. Staining was prevalent in almost all cellular compartments of the tonsil.

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  Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Western blot staining of HeLa whole cell lysate using rabbit Anti-HDAC1 antibody

  All lanes: Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845) at 1 µg/mL

  Lane 1: HeLa whole cell lysate at 20 µg

  Lane 2: HeLa whole cell lysate at 20 µg with Human HDAC1 peptide (ab20434)

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (Goat Anti-Rabbit IgG H&L (HRP) preadsorbed ab7090) at 1/5000 dilution

  Predicted band size: 55 kDa

  Observed band size: 60 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-HDAC1 antibody

  ab19845 at a 1/3000 dilution staining asynchronous HeLa cells by ICC/IF. The cells were paraformaldehyde fixed and immunofluorescently labelled with ab19845 for 30 minutes at room temperature. Bound antibody was detected using a Cy3 conjugated goat anti-rabbit antibody. Nuclei were visuallised using DAPI staining. The antibody was found to be highly enriched in the nucleus.

  This image is courtesy of an Abreview submitted by Kirk McManus.

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  Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-HDAC1 antibody

  ICC/IF image of ab19845 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab19845, 1µg/ml) overnight at +4°C. 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 4% PFA fixed (10 min) HepG2 cells at 1µg/ml, and in 100% methanol fixed (5 min) MCF7 and HepG2 cells at 1µg/ml

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-HDAC1 antibody

  IHC image of HDAC1 staining in human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab19845, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-HDAC1 antibody - Nuclear Loading Control (ab19845)

  HDAC1 Western blot staining using rabbit Anti-HDAC1 antibody

  HDAC1 western blot using anti-HDAC1 antibody ab19845. Publication image and figure legend from Ma, C., Wang, F., et al., 2018, Mol Cancer, PubMed 29625565.

  
  

  ab19845 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab19845 please see the product overview.

  Involvement of NuRD complex in the regulation of breast cancer cell senescence and suppression mediated by SALL1. a and b Transfection of mutated SALL1 (mSALL1, deleted the NuRD binding peptide motif of conserved 12-amino) in MCF-7 and E0771 cancer cells did not induce SA-β-Gal+ cell populations (in a) and promote cancer cell cycle arrest in S phase (in b). In contrast, transfection of full-length SALL1 into MCF-7 and E0771 breast cancer cells significantly induced tumor cell senescence (around 40%) and promoted cell cycle arrest in S phase. Breast cancer cells were transfected with the indicated constructs and cultured for additional 72 h. Senescent cells were analyzed using the SA-β-Gal activity assay and the cell cycle distribution in tumor cells was analyzed after incubation with propidium iodide. Data shown in (a) are mean ± SD from three independent experiments with similar results. **p < 0.01 compared with the mSALL1 and vector control groups. c and d Transfection of SALL1-S2E into MCF-7 and E0771 breast cancer cells lost the ability to induce tumor cell senescence. However, transfection of SALL1-S2A into breast cancer cells significantly augmented senescence induction in both cell lines compared with that of in wild type SALL1-transfected tumor cells. Cell transfection procedure and SA-β-Gal+ cell determination were identical to (a). SALL1-S2E: substitution of the serine with a glutamic acid in SALL1. SALL1-S2A: mutating the serine to an alanine in SALL1. SA-β-Gal+ tumor cells were identified with dark blue granules as indicated by the arrows (in c). Data shown in (d) are mean ± SD from three independent experiments with similar results. **p < 0.01, compared with the vector control group. #p < 0.01, compared with the wild type SALL1 group. e Transfection of wild type SALL1 and SALL1-S2A into MCF-7 tumor cells recruited NuRD complex components determined with GST pulldown analyses. In contrast, transfection of SALL1-S2E markedly disrupted recruitment of NuRD components. MCF-7 cells were transfected with or without plasmids pEBG-SALL1, pEBG-SALL1-S2A, and pEBG-SALL1-S2E, and cultured for 3 days. Total protein lysates precipitated with Protein G-Sepharose beads. Pulldowns were analyzed by western blot with antibodies against SALL1, HDAC1, MTA2, MBD3 and RbAp46/48