Anti-Daxx antibody [E94]

10 product images

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  Lanes 1- 2: Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab32140 was shown to react with Daxx in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human DAXX knockout HeLa cell line ab265233 (knockout cell lysate Human DAXX knockout HeLa cell lysate ab257408) was used. Wild-type HeLa and DAXX knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32140 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 5000 dilution and a 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.

  All lanes: Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: DAXX knockout HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human DAXX knockout HeLa cell line (Human DAXX knockout HeLa cell line ab265233)

  Performed under reducing conditions.

  Predicted band size: 81 kDa

  Observed band size: 100 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Daxx antibody [E94] (ab32140)

  ab32140 stained HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32140 at 10μg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  Lanes 1 - 2: Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - loading control, Anti-GAPDH antibody [mAbcam 9484] - Loading Control ab9484, observed at 37 kDa.
  

  
  ab32140 was shown to specifically react with Daxx in wild-type HAP1 cells as signal was lost in DAXX knockout cells. Wild-type and DAXX knockout samples were subjected to SDS-PAGE. ab32140 and Anti-GAPDH antibody [mAbcam 9484] - Loading Control ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Daxx antibody [E94] (ab32140)

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

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

  Predicted band size: 81 kDa

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  Blocking and diluting buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Daxx antibody [E94] (ab32140) at 0.271 µg/mL

  Lane 1: PC-12(Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

  Lane 2: NIH/3T3(Mouse embryonic fibroblast) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 81 kDa

  Exposure time: 29s

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  Flow Cytometry (Intracellular) - Anti-Daxx antibody [E94] (ab32140)

  Overlay histogram showing HeLa cells stained with ab32140 (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 (ab32140, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Daxx antibody [E94] (ab32140)

  ab32140, at a dilution of 1/50, staining Daxx in paraffin embedded human stomach adenocarcinoma tissue by Immunohistochemistry.
  

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  All lanes: Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

  All lanes: Hela cell lysate

  Predicted band size: 81 kDa

  Observed band size: 100 kDa, 48 kDa

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  False colour image of Western blot: Anti-Daxx antibody [E94] staining at 1/5000 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, ab32140 was shown to bind specifically to Daxx. A band was observed at 105 kDa in wild-type MCF7 cell lysates with no signal observed at this size in DAXX knockout cell line. To generate this image, wild-type and DAXX knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Daxx antibody [E94] (ab32140) at 1/5000 dilution

  Lane 1: Wild-type MCF7 cell lysate at 20 µg

  Lane 2: DAXX knockout MCF7 cell lysate at 20 µg

  Lane 3: THP-1 cell lysate at 20 µg

  Lane 4: K562 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: 81 kDa

  Observed band size: 105 kDa

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  False colour image of Western blot: Anti-Daxx antibody [E94] staining at 1/5000 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, ab32140 was shown to bind specifically to Daxx. A band was observed at 105 kDa in wild-type A549 cell lysates with no signal observed at this size in DAXX knockout cell line.
  
  To generate this image, wild-type and DAXX 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.

  Lanes 1 - 3: Western blot - Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

  Lanes 1 - 3: Western blot - Anti-Daxx antibody [E94] - BSA and Azide free (Anti-Daxx antibody [E94] - BSA and Azide free ab239806) at 1/5000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: Western blot - Human DAXX knockout A549 cell line (Human DAXX knockout A549 cell line ab287356)

  Lane 2: DAXX knockout A549 cell lysate at 20 µg

  Lane 3: THP-1 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: 105 kDa

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  Western blot - Anti-Daxx antibody [E94] (ab32140)

  False colour image of Western blot: Anti-Daxx antibody [E94] staining at 1/5000 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, ab32140 was shown to bind specifically to Daxx. A band was observed at 100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in DAXX knockout cell line. To generate this image, wild-type and DAXX 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-Daxx antibody [E94] (ab32140) at 1/5000 dilution

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

  Lane 2: Western blot - Human DAXX knockout HCT116 cell line (Human DAXX knockout HCT116 cell line ab287355)

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

  Lane 3: THP-1 cell lysate at 20 µg

  Lane 4: K562 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: 81 kDa

  Observed band size: 100 kDa