Anti-IDH2 antibody [EPR7576]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IDH2 antibody [EPR7576] (AB129180)

Flow cytometry overlay histogram showing wild-type Jurkat (green line) and IDH2 knockout Jurkat stained with ab129180 (magenta line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab129180) (1x 10⁶ in 100μl at 0.008 μg/ml (1/111625)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control in Jurkat WT cells (black line) and Jurkat-IDH2 KO cells (grey line), used at the same concentration and conditions as the primary antibody.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• Flow Cyt (Intra)

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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) (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.

• IHC-P

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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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IDH2 antibody [EPR7576] (AB129180)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat IDH2 knockout cells labeling IDH2 with ab129180 at 0.2 μg/ml. Cells were counterstained with ab7291 Anti-alpha Tubulin antibody [DM1A] at 1 : 1000 (1 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488 ab150081, green) and goat anti mouse IgG (Alexa Fluor® 594 ab150120, magenta) were used as the secondary antibodies at 1 : 1000 (2 μg/ml) dilution. The nuclear counterstain was DAPI (Blue).

• WB

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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

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• WB

Lab

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, 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 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 (ab216773) and 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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• WB

Lab

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] (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 <a href='/en-us/products/cell-lines/human-idh2-knockout-jurkat-cell-line-ab282331'>ab282331</a> 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

Observed band size: 51 kDa

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• WB

Lab

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] (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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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 2:

Western blot - Human IDH2 knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-idh2-knockout-jurkat-cell-line-ab282331'>ab282331</a>)

Lane 3:

MOLT-4 cell lysate at 20 µg

Lane 4:

HEK-293 cell lysate at 20 µg

Predicted band size: 50 kDa

Observed band size: 48 kDa

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