Anti-IDH2 antibody [EPR7576]

6 product images

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  Western blot - Anti-IDH2 antibody [EPR7576] (ab129180)

  False colour image of Western blot: Anti-IDH2 antibody [EPR7576] 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, ab129180 was shown to bind specifically to IDH2. A band was observed at 48 kDa in wild-type Jurkat cell lysates with no signal observed at this size in IDH2 knockout cell line Human IDH2 knockout Jurkat cell line ab282331 (knockout cell lysate ab283148). To generate this image, wild-type and IDH2 knockout Jurkat 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 (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) at 1/20000 dilution.

  All lanes: Western blot - Anti-IDH2 antibody [EPR7576] (ab129180) at 1/1000 dilution

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

  Lane 2: IDH2 knockout Jurkat cell lysate at 20 µg

  Lane 3: MOLT-4 cell lysate at 20 µg

  Lane 4: HEK-293 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 50 kDa

  Observed band size: 48 kDa

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  Western blot - Anti-IDH2 antibody [EPR7576] (ab129180)

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: IDH2 knockout HAP1 cell lysate (20 μg)
  Lane 3: K562 cell lysate (20 μg)
  Lane 4: HepG2 cell lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab129180 observed at 47 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  ab129180 was shown to recognize IDH2 when IDH2 knockout samples were used, along with additional cross-reactive bands. Wild-type and IDH2 knockout samples were subjected to SDS-PAGE. ab129180 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-IDH2 antibody [EPR7576] (ab129180)

  Predicted band size: 50 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH2 antibody [EPR7576] (ab129180)

  ab129180, at 1/250 dilution, staining IDH2 in Formalin-fixed, Paraffin-embedded Human thyroid gland carcinoma by Immunohistochemistry.
  

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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  Flow Cytometry (Intracellular) - Anti-IDH2 antibody [EPR7576] (ab129180)

  Overlay histogram showing MCF7 cells stained with ab129180 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab129180, 1/10000 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) (0.1μg/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. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

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  Western blot - Anti-IDH2 antibody [EPR7576] (ab129180)

  All lanes: Western blot - Anti-IDH2 antibody [EPR7576] (ab129180) at 1/1000 dilution

  Lane 1: Molt-4 cell lysate at 10 µg

  Lane 2: K562 cell lysate at 10 µg

  Lane 3: 293T cell lysate at 10 µg

  Lane 4: HepG2 cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-rabbit HRP

  Predicted band size: 50 kDa

  Observed band size: 45 kDa

• 

  Western blot - Anti-IDH2 antibody [EPR7576] (ab129180)

  Western blot: Anti-IDH2 antibody [EPR7576] (ab129180) 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 magenta. In Western blot, ab129180 was shown to bind specifically to IDH2. A band was observed at 51 kDa in wild-type A549 cell lysates with no signal observed at this size in IDH2 knockout cell line. To generate this image, wild-type and IDH2 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-IDH2 antibody [EPR7576] (ab129180) at 1/1000 dilution

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

  Lane 2: IDH2 knockout A549 cell lysate at 20 µg

  Lane 3: Wild-type Jurkat cell lysate at 20 µg

  Lane 4: IDH2 knockout Jurkat Human IDH2 knockout Jurkat cell line ab282331 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: 51 kDa