Anti-IDH2 antibody [5F11]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH2 antibody [5F11] (AB55271)

IDH2 antibody (ab55271) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human colon.

This image was generated using the ascites version of the product.

• Flow Cyt

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Flow Cytometry - Anti-IDH2 antibody [5F11] (AB55271)

Overlay histogram showing MCF7 cells stained with ab55271 (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 (ab55271, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. 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.

This image was generated using the ascites version of the product.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IDH2 antibody [5F11] (AB55271)

ICC/IF image of ab55271 stained Mcf7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab55271, 10μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

This image was generated using the ascites version of the product.

• WB

Lab

Western blot - Anti-IDH2 antibody [5F11] (AB55271)

False colour image of Western blot : Anti-IDH2 antibody [5F11] staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab55271 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-IDH2 antibody [5F11] (ab55271) 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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• WB

Lab

Western blot - Anti-IDH2 antibody [5F11] (AB55271)

Western blot : Anti-IDH2 antibody [5F11] (ab55271) staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab55271 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IDH2 antibody [5F11] (ab55271) 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 51 kDa

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• WB

Unknown

Western blot - Anti-IDH2 antibody [5F11] (AB55271)

Western blot against tagged recombinant protein immunogen using ab55271 IDH2 antibody at 1ug/ml. Predicted band size of immunogen is 37 kDa

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-IDH2 antibody [5F11] (ab55271)

Predicted band size: 50 kDa

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• WB

Unknown

Western blot - Anti-IDH2 antibody [5F11] (AB55271)

The blocking agent used is 5% milk.

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-IDH2 antibody [5F11] (ab55271)

Lane 1:

IDH2 in transfected 293T cell line

Lane 2:

Non-transfected lysate

Predicted band size: 50 kDa

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• WB

CiteAb

Western blot - Anti-IDH2 antibody [5F11] (AB55271)

Western Blotting using Anti-IDH2 antibody [5F11], ab55271. Publication image from Lee, W. D. et al., 2019, Nat Commun, 30903027. Legend direct from paper.

A quantitative view of mitochondrial and cytosolic fluxes in the TCA cycle and citrate metabolism under normoxia. a–d Mass-isotopomer labeling kinetics of citrate in mitochondria (a) and cytosol (b), and malate in mitochondria (c) and cytosol (d) when feeding HeLa cells with [U-13C]-glutamine under standard normoxic conditions. e Measured isotopic labeling ratio in HeLa cells for citrate m + 5 /m + 4 and malate m + 3 /m + 4 in media (red) in comparison to the expected labeling via computational simulation, considering the measured labeling kinetics of citrate and malate in mitochondria (green) and cytosol (orange). f Gibbs free energy of mitochondrial (IDH2/3) and cytosolic (IDH1) IDH isozymes (in the oxidative direction) in HeLa cells under normoxia (green) and hypoxia (red). g Mitochondrial and cytosolic fluxes, showing percentage from citrate synthase flux (which is 0.48 mM h−1). Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes are shown in Supplementary Data 8. h The measured mass-isotopomer distribution of palmitate when feeding cells with [U-13C]-glutamine (green) is consistent with the simulated fit (orange). For the simulation, acetyl-CoA labeling was assumed to follow a binomial distribution with a probability of 7.1% having m + 2 labeling form. i Validation of the method based on knock-down of IDH1 or IDH2 genes and following citrate isotopic labeling after feeding cells with [U-13C]-glutamine. Upon IDH1 knockdown, the ratios between citrate m + 5 and m + 4 in mitochondria and cytosol are similar, indicating that all reductive glutamine flux occurred in mitochondria (where citrate m + 4 is produced from malate m + 4). Meanwhile, IDH2 knockdown resulted in a higher citrate m + 5 to m + 4 ratio in cytosol, indicating that reductive IDH1 remains active. n.s. not significant. *P < 0.05 and **P < 0.01 by two-sample t-test. Data are mean ± SD, n = 3 independent biological replicates

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