Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade

8 product images

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  ChIP - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Chromatin was prepared from K-562 cells according to theAbcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
  The ChIP was performed with 25 μg of chromatin, 5 μg of ab275378 (red), or 5 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 25 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

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  Immunoprecipitation - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  KAT3B/p300 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab275378 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275378 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  

  Lane 1: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug

  Lane 2: ab275378 IP in K-562 whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab275378 in K-562 whole cell lysate

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 76 seconds.

  Lysate was freshly prepared and used in IP immediately to mininize protein degradation.

  Incubation time was 2 hours.

  All lanes: Immunoprecipitation - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Predicted band size: 264 kDa

  Observed band size: 300 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling KAT3B/p300 with ab275378 at 1/100 (4.77 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in NIH/3T3 cell line. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 dilution (Red). The Nuclear counterstain was DAPI (Blue).
  

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

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  Flow Cytometry (Intracellular) - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling KAT3B/p300 with ab275378 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
  

  A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.

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  Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  Both recombinant proteins were expressed from a E.coil expression system. Sample loaded onto lane 2 was E.coil extract containing CREBBP recombinant protein.

  This product has weak cross reaction with CREBBP protein.

  Exposure time: 5.5 seconds.

  All lanes: Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378) at 1/1000 dilution

  Lane 1: His-tagged human KAT3B/p300 recombinant protein 20 ng

  Lane 2: His-tagged human CREBBP recombinant protein, 20 ng

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 264 kDa

  Observed band size: 88.7 kDa, 46.3 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling KAT3B/p300 with ab275378 at 1/100 (4.77 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in HeLa cell line. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
  

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

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  Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  The molecular weight observed is consistent with what has been described in the literature (PMID: 8995708).

  Lysates were made freshly and used in WB immediately to minimize protein degradation.

  Exposure times: Lane 1: 125 seconds;Lanes 2-3: 3 minutes.

  All lanes: Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378) at 1/1000 dilution

  Lane 1: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

  Lane 2: RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

  Lane 3: PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 264 kDa

  Observed band size: 300 kDa

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  Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378)

  Western blot: Anti-EP300 antibody [EPR23495-268] (ab275378) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab275378 was shown to bind specifically to EP300. A band was observed at 200/90-130 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EP300 knockout cell line. To generate this image, wild-type and EP300 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378) at 1/1000 dilution

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: EP300 knockout HCT 116 cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: Human brain olfactory lobe cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 200 kDa, 90-130 kDa