Anti-Histone H4 (di methyl K20) antibody

5 product images

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  Immunoprecipitation - Anti-Histone H4 (di methyl K20) antibody (ab9052)

  Histone H4 (di methyl K20) was immunoprecipitated using 0.5mg HeLa whole cell extract, 5μg of Rabbit polyclonal to Histone H4 (di methyl K20) 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 ab9052.
  Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
  Band: 14kDa: Histone H4 (di methyl K20).

  All lanes: Immunoprecipitation - Anti-Histone H4 (di methyl K20) antibody (ab9052)

  Predicted band size: 11 kDa

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  This image is courtesy of Steve Sanders, Tony Kouzarides lab, University of Cambridge

  Western blot - Anti-Histone H4 (di methyl K20) antibody (ab9052)

  All lanes: Western blot - Anti-Histone H4 (di methyl K20) antibody (ab9052) at 1/2000 dilution

  Performed under reducing conditions.

  Predicted band size: 11 kDa

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  Western blot - Anti-Histone H4 (di methyl K20) antibody (ab9052)

  Di methylation at K20 is seen in S. pombe and in calf thymus (Roche). S. cerevisiae lacks K20 (di) methylation.

  All lanes: Upper blot - Histone H4 antibody (gift of A. Verreault) Lower blot - Rabbit polyclonal to Histone H4 di methyl K20 (ab9052) at 1/2000

  Lane 1: S.cerevisiae extract

  Lane 2: S.pombe extract

  Lane 3: S.pombe rH4

  Lane 4: Drosophila rH4

  Lane 5: Histones (Roche)

  Predicted band size: 11 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (di methyl K20) antibody (ab9052)

  IHC image of ab9052 staining in human pancreas adenocarcinoma 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 ab9052, 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.
  
  
  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H4 (di methyl K20) antibody (ab9052)

  ab9052 staining Histone H4 (di methyl K20) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab9052 at 0.1µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

  Also suitable in cells fixed with 4% paraformaldehyde (10 min).

  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.