Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade

5 product images

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  Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165)

  Histone H2B was immunoprecipitated using 0.5mg HeLa whole cell extract, 5μg of Mouse monoclonal to Histone H2B 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°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab64165.

  Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

  Band: 17kDa; Histone H2B

  All lanes: Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165)

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 14 kDa

  Exposure time: 20min

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  ChIP - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165)

  Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 5μg of ab64165 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

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  Western blot - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165)

  All lanes: Western blot - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165) at 5 µg/mL

  Lane 1: Histone H1 recombinant protein. at 0.1 µg/mL

  Lane 2: Histone H2A recombinant protein. at 0.1 µg/mL

  Lane 3: Histone H2B recombinant protein. at 0.1 µg/mL

  Lane 4: Histone H3 recombinant protein. at 0.1 µg/mL

  Lane 5: Histone H4 recombinant protein. at 0.1 µg/mL

  Secondary

  All lanes: Rabbit polyclonal to Mouse IgG - H&L (HRP) at 1/3000 dilution

  Performed under reducing conditions.

  Predicted band size: 14 kDa

  Observed band size: 17 kDa

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  Flow Cytometry (Intracellular) - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165)

  Overlay histogram showing HeLa cells stained with ab64165 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab64165, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (Mouse IgG2b [PLPV219] - Isotype Control ab91366, 2µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165)

  ab64165 staining Histone H2B 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 ab64165 at 1µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

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