Anti-NDUFB6 antibody [21C11BC11]

• Flow Cyt

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Flow Cytometry - Anti-NDUFB6 antibody [21C11BC11] (AB110244)

Overlay histogram showing HepG2 cells stained with ab110244 (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. The cells were then incubated with the antibody (ab110244, 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 IgG2b [PLPV219] (ab91366, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• WB

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Western blot - Anti-NDUFB6 antibody [21C11BC11] (AB110244)

All lanes:

Western blot - Anti-NDUFB6 antibody [21C11BC11] (ab110244) at 0.5 µg/mL

Lane 1:

isolated mitochondria from Human heart at 5 µg

Lane 2:

isolated mitochondria from cow heart at 1 µg

Lane 3:

isolated mitochondria from rat heart at 10 µg

Lane 4:

isolated mitochondria from mouse heart at 10 µg

Predicted band size: 15 kDa

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• WB

CiteAb

Western blot - Anti-NDUFB6 antibody [21C11BC11] (AB110244)

NDUFB6 western blot using anti-NDUFB6 antibody [21C11BC11] ab110244. Publication image and figure legend from Beutner, G., Eliseev, R. A., et al., 2014, PLoS One, PubMed 25427064.

ab110244 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 ab110244 please see the product overview.

The assembly of ETC complexes into supercomplexes begins around E11.5.A. One-dimensional high resolution clear native PAGE (hrCN-PAGE) shows the presence of monomeric Cx-1 and the appearance of Cx-1 containing supercomplexes during embryonic development and in adult mouse hearts. Whole tissue homogenates (10 µg for embryonic samples and 5 µg for adult sample) from hearts were solubilized with digitonin (4g digitonin/g protein) and separated by 5–15% hrCN-PAGE. The proteins were then transferred onto nitrocellulose membranes and Cx-1 was visualized first by the detection of NDUFAB1 (ab1, a rabbit polyclonal antibody). Then, the membrane was stripped and re-probed against NDUFB6 (B6, a mouse-monoclonal antibody). In addition, an in-gel assay of Cx-1 (IGA) demonstrates functional complex I monomers and supercomplexes are present in adult heart homogenates. B and C. Quantitative analysis of the presence of monomeric Cx-1 (B) and supercomplexes containing Cx-1 (C). NS–not significant, *p≤0.05, **p≤0.01, ***p≤0.005, and ****p≤0.0001 compared to adult heart samples using ANOVA with Tukey post hoc testing; n = 5. D. Immunocapture of supercomplexes using anti-Cx-3 antibodies; the immunoprecipitate was analyzed by SDS PAGE/immunoblot for the presence of subunits of Cx-1 (NDUFB6 and NDUFA9), Cx-3 (ubiquinol-cytochrome reductase core protein 1 precursor, Core 1) and Cx-4 (cytochrome c oxidase, subunit 4 (Cox 4)). Homogenates from E9.5, E11.5, and 13.5 heart tissue and homogenate (TH) and mitochondria (M) from adult hearts were examined. Representative immunoblots from n = 3, although these immunoblots are overexposed to demonstrate labeling at younger ages.

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