Anti-KDM6A / UTX antibody [EPR23203-211]

5 product images

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  Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183)

  Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
  

  Lanes 1-4: Merged signal (red and green). Green - ab253183 observed at 160 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

  ab253183 Anti-KDM6A antibody [EPR23203-211] was shown to react with KDM6A in HAP1 wild-type cells in Western blot. Loss of signal was observed when KDM6A knockout sample was used. Wild-type and KDM6A knockout samples were subjected to SDS-PAGE.

  ab253183 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated at room temperature for 2. 5 hours at 1/1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

  Negative control: THP-1 (PMID: 19330029).

  The expression profile and molecular weight observed is consistent with what has been described in the literature (PMID: 19330029).

  The the bands between 50kDa and 150kDa are caused by degradation based on the result of fresh lysates.

  All lanes: Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183) at 1/1000 dilution

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: KDM6A knockout HAP1 whole cell lysate at 20 µg

  Lane 3: F9 (mouse embryonal carcinoma epithelial cell), whole cell lysate at 20 µg

  Lane 4: THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Predicted band size: 154 kDa

  Observed band size: 160 kDa

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  Flow Cytometry (Intracellular) - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183)

  Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte), (Left panel) / HAP1 (Human chronic myelogenous leukemia near-haploid cell line), (Right panel) cells labelling KDM6A / UTX with ab253183 at 1/50 dilution (1μg) (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.

  Negative control: THP-1.

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  Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183)

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

  Fresh lysates were used in this WB.

  Exposure time: 3 minutes.

  All lanes: Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183) at 1/1000 dilution

  Lane 1: HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

  Lane 2: NIH/3T3 (mouse embryonic fibroblast), 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: 154 kDa

  Observed band size: 160 kDa

• 

  Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183)

  Western blot: Anti-KDM6A antibody [EPR23203-211] (ab253183) 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, ab253183 was shown to bind specifically to KDM6A. A band was observed at 150-160 kDa in wild-type A549 cell lysates with no signal observed at this size in KDM6A knockout cell line. To generate this image, wild-type and KDM6A knockout A549 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-KDM6A / UTX antibody [EPR23203-211] (ab253183) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 7 µg

  Lane 2: KDM6A knockout A549 cell lysate at 7 µg

  Lane 3: Wild-type HAP1 cell lysate at 7 µg

  Lane 4: KDM6A knockout HAP1 cell lysate at 7 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: 154 kDa

  Observed band size: 150 kDa, 160 kDa

• 

  Flow Cytometry (Intracellular) - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183)

  Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized F9 (Mouse embryonal carcinoma epithelial cell) cells labelling KDM6A / UTX with ab253183 at 1/500 dilution (0.1μg) (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.