Anti-IDH2 antibody [EPR7577]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IDH2 antibody [EPR7577] (AB131263)

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling IDH2 with Purified ab131263 at 1 : 1000 dilution (0.2 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IDH2 antibody [EPR7577] (AB131263)

Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labelling IDH2 with Purified ab131263 at 1 : 20 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1 : 2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH2 antibody [EPR7577] (AB131263)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue sections labeling IDH2 with Purified ab131263 at 1 : 100 (2.11 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IDH2 antibody [EPR7577] (AB131263)

Flow cytometry overlay histogram showing wild-type Jurkat (green line) and IDH2 knockout Jurkat stained with ab131263 (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 (ab131263) (1x 10⁶ in 100μl at 0.008 μg/ml (1/27625)) 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 530/30 bandpass filter.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IDH2 antibody [EPR7577] (AB131263)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat IDH2 knockout cells labeling IDH2 with ab131263 at 1 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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH2 antibody [EPR7577] (AB131263)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue sections labeling IDH2 with Purified ab131263 at 1 : 100 (2.11 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH2 antibody [EPR7577] (AB131263)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labeling IDH2 with Purified ab131263 at 1 : 100 (2.11 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• WB

Lab

Western blot - Anti-IDH2 antibody [EPR7577] (AB131263)

Western blot : Anti-IDH2 antibody [EPR7577] (ab131263) 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, ab131263 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 [EPR7577] (ab131263) 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 [EPR7577] (AB131263)

False colour image of Western blot : Anti-IDH2 antibody [EPR7577] 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, ab131263 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 [EPR7577] (ab131263) 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>)

Predicted band size: 50 kDa

Observed band size: 48 kDa

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• WB

Lab

Western blot - Anti-IDH2 antibody [EPR7577] (AB131263)

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 - ab131263 observed at 48 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab131263 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. ab131263 and ab8245 (loading control to GAPDH) were both diluted 1/10 000 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 [EPR7577] (ab131263)

Predicted band size: 50 kDa

false

• WB

Unknown

Western blot - Anti-IDH2 antibody [EPR7577] (AB131263)

All lanes:

Western blot - Anti-IDH2 antibody [EPR7577] (ab131263) at 1/5000 dilution

Lane 1:

MOLT-4 (Human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 3:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

Mouse liver lysate at 20 µg

Lane 6:

Rat liver lysate at 20 µg

Lane 7:

Mouse kidney lysate at 20 µg

Lane 8:

Rat kidney lysate at 20 µg

Lane 9:

Rat stomach lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 50 kDa

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