Anti-NDUFS2 antibody [7A12BE5AD5]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

Mitochondrial localization of Complex I subunit NDUFS2 visualized by Immunocytochemistry in HDFn cultured cells (normal Human dermal fibroblasts, neonatal), using ab110249 at 5 μg/ml.

• Flow Cyt

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Flow Cytometry - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

Overlay histogram showing HepG2 cells stained with ab110249 (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 (ab110249, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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.

• WB

Lab

Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

ab110249 was shown to react with NDUFS2 in wild-type HAP1 cells in Western blot with loss of signal observed in a NDUFS2 knockout cell line. Wild-type HAP1 and NDUFS2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab110249 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (ab110249) at 1/5000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

NDUFS2 knock-out HAP1 lysate at 20 µg

Predicted band size: 53 kDa

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• WB

Lab

Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : NDUFS2 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : Jurkat whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab110249 observed at 48 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab110249 was shown to specifically react with NDUFS2 in wild-type HAP1 cells whilst signal was lost in NDUFS2 knockout cells. Wild-type and NDUFS2 knockout samples were subjected to SDS-PAGE. ab110249 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 0.25 μg/ml and 1/20,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (ab110249)

Predicted band size: 53 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

ab110249 staining NDUFS2 in wild-type Hap1 cells (top panel) and NDUFS2 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab110249 at 0.4μg/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor^® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• WB

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Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

Complex I-subunit NDUFS2 is detected in the Human samples with Human specific ab110249, while Complex I-subunit NDUFB8 is detected in all samples (Human, Mouse and Rat) with an anti-NDUFB8.

All lanes:

Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (ab110249) at 0.25 µg/mL

Lane 1:

Molecular Weight Markers

Lane 2:

Isolated mitochondria from Human liver

Lane 3:

Isolated mitochondria from Bovine heart

Lane 4:

Isolated mitochondria from H9C2 cells (Rat cardiomyocyte)

Lane 5:

Isolated mitochondria from MEF cells (Mouse embryo fibroblast)

Lane 6:

Isolated mitochondria from HepG2

Predicted band size: 53 kDa

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• WB

CiteAb

Western blot - Anti-NDUFS2 antibody [7A12BE5AD5] (AB110249)

NDUFS2 western blot using anti-NDUFS2 antibody [7A12BE5AD5] ab110249. Publication image and figure legend from Hernansanz-Agustín, P., Ramos, E., et al., 2017, Redox Biol, PubMed 28511347.

ab110249 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab110249 please see the product overview.

Silencing of complex I subunits specifically affects the assembly of complex I-containing supercomplexes (a and b) Protein extracts from BAECs treated with siSCR, siNDUFS4 or siNDUFS2 were immunoblotted for NDUFS4 or NDUFS2 proteins with tubulin as loading control. Up : representative image; down : quantification of three independent experiments (mean±s.e.m.). (c and d) BN-PAGE of siSCR-treated or siNDUFS4-treated BAECs, analyzed by western blot with antibodies against NDUFB6 (complex I; c) or Core I (complex III; d). Representative image of three independent experiments.

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