Anti-KAT2A / GCN5 antibody [EPR21146] - BSA and Azide free

4 product images

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  ChIP - Anti-KAT2A / GCN5 antibody [EPR21146] - BSA and Azide free (ab231075)

  Chromatin was prepared from A549 treated with thapsigargin (1μM 12 hours) cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
  The ChIP was performed with 25 μg of chromatin, 5 μg of Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 (red), and 20 μl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  

  Primers and probes are located in the first kb of the transcribed region.
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876).

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  Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - BSA and Azide free (ab231075)

  Blocking/Dilution buffer: 5% NFDM/TBST
  

  Exposure time: 3 minutes

  Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 was shown to specifically react with KAT2A in wild type HAP1 cells. No band was observed when KAT2A knockout samples were used. Wild-type and KAT2A knockout samples were subjected to SDS-PAGE. Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/5000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876).

  All lanes: Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - BSA and Azide free (ab231075) at 1/1000 dilution

  Lane 1: Wild type HAP1 whole cell lysate at 10 µg

  Lane 2: KAT2A / GCN5-knockout HAP1 whole cell lysate whole cell lysate at 10 µg

  Lane 3: HEK-293T (uman epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 10 µg

  Lane 4: MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Predicted band size: 94 kDa

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  Immunoprecipitation - Anti-KAT2A / GCN5 antibody [EPR21146] - BSA and Azide free (ab231075)

  KAT2A / GCN5 was immunoprecipitated from 0.35mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 at 1/30 dilution. Western blot was performed from the immunoprecipitate using Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/5000 dilution.
  

  Lane 1: HeLa whole cell lysate 10ug (Input).

  Lane 2: Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 IP in HeLa whole cell lysate.

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 in HeLa whole cell lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  Exposure time: 30 seconds.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876).

  All lanes: Immunoprecipitation - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876)

  Predicted band size: 94 kDa

  Observed band size: 94 kDa

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  Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - BSA and Azide free (ab231075)

  False colour image of Western blot: Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876 was shown to bind specifically to KAT2A / GCN5. A band was observed at 95 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in kat2a knockout cell line. To generate this image, wild-type and kat2a knockout U-2 OS 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.
  
  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876).

  All lanes: Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade ab217876) at 1/1000 dilution

  Lane 1: Wild-type U-2 OS cell lysate at 20 µg

  Lane 2: GCN5 knockout U-2 OS cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: NIH/3T3 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.

  Predicted band size: 94 kDa

  Observed band size: 95 kDa