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Total OXPHOS Rodent WB Antibody Cocktail (ab110413) is part of the reagents, controls & accessories range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
WB
NDUFB8
Mouse, Rat, Cow, Human, Cynomolgus monkey
Application | Reactivity | Dilution info | Notes |
---|---|---|---|
Application WB | Reactivity Reacts | Dilution info - | Notes The antibody cocktail (1.5 mg/mL) should be diluted 250x to a final working concentration of 6.0 µg/mL for Western blotting. We recommend using PBS with 1% milk as the antibody diluent. Store the antibody cocktail at 4°C and the control sample at -80°C. We recommend using a high pH CAPS / PVDF transfer protocol when using this antibody for Western blot. Please contact us for more details. |
Accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), that is believed not to be involved in catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone.
Complex I-ASHI, NADH-ubiquinone oxidoreductase ASHI subunit, CI-ASHI, NDUFB8
Total OXPHOS Rodent WB Antibody Cocktail (ab110413) is part of the reagents, controls & accessories range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Complex I-ASHI, NADH-ubiquinone oxidoreductase ASHI subunit, CI-ASHI, NDUFB8
WB
NDUFB8
Mouse, Rat, Cow, Human, Cynomolgus monkey
Dry Ice
Multi
Total OXPHOS Rodent WB Antibody Cocktail ab110413 is an optimized cocktail of high quality antibodies for analyzing relative levels of OXPHOS complexes in rat or mouse mitochondria by western blot.
This OXPHOS cocktail contains 5 mouse mAbs, one each against CI subunit NDUFB8 (ab110242), CII-30kDa (ab14714), CIII-Core protein 2 (ab14745), CIV subunit I (ab14705) and CV alpha subunit (ab14748) as an optimized premixed cocktail. The kit is suitable for Western Blotting analysis of the relative levels of the 5 OXPHOS complexes in mitochondrial preparations from mouse, rat, human, or bovine sources. The positive control supplied with the cocktail is ab110341.
Altered levels of assembly can arise from mutations in individual subunits, mutations in assembly factors for the complex(es), mtDNA depletion or as a result of physiological and or pathological changes e.g. hormone treatment, exercise, diet or oxidative stress.
The mAbs in the cocktail were chosen because they are each against a subunit that is labile when its complex is not assembled. Also, the different subunits are easily resolved in SDS-PAGE (see protocols).
Note: Mouse tissue samples can easily be contaminated with antibodies from the animal's blood. To avoid background bands, use ab110413 with an anti-mouse secondary against native antibodies. Also, COXI is a highly hydrophobic protein and appears as a broad band at ~35 kDa (not at its true molecular weight at 57 kDa). It is very sensitive to heating. Therefore, the samples, including the positive control, should not be heated over 50°C before loaded on the gel.
The Western blot cocktail is supplied at a concentration of 1.5 mg/mL.
We recommend using a high pH CAPS / PVDF transfer protocol when using this antibody for Western blot.
Store the antibody cocktail at 4°C and the control sample at -80°C.
**Related products** Review the , or the full to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.
OXPHOS proteins are integral to the production of ATP the primary energy molecule in cells. They form a multi-protein complex that transduces energy by transferring electrons and pumping protons across the inner mitochondrial membrane. The OXPHOS system comprises complexes I to V each playing a specific role within this energy-converting process. This system is important for cellular respiration acting as the powerhouse that drives numerous cellular processes.
MitoProfile® Total OXPHOS Rodent refers to a collection of proteins associated with oxidative phosphorylation (OXPHOS) in rodents. This target includes several key protein complexes involved in the electron transport chain within mitochondria. These complexes specified as OXPHOS Complexes I-V act in conjunction to facilitate ATP production. OXPHOS proteins such as NADH dehydrogenase (complex I) cytochrome c oxidase (complex IV) and ATP synthase (complex V) are detected using antibodies specified in a mitochondrial cocktail or OXPHOS cocktail tailored for Western blots. Mitochondria express these proteins across various tissues including skeletal muscle heart and brain reflecting their essential role in energy metabolism.
OXPHOS proteins participate significantly in the mitochondrial electron transport chain and oxidative phosphorylation pathways. The ATP produced through this mechanism contributes to various metabolic pathways and cellular activities. Cytochrome c associated with complex IV plays a pivotal role in apoptotic pathways linking energy metabolism with cell death processes. Other key proteins such as ubiquinone function as electron carriers in the electron transport chain to ensure efficient ATP synthesis.
Impaired OXPHOS function associates with metabolic disorders such as mitochondrial diseases and neurodegenerative conditions like Parkinson's disease. Defects in any of the OXPHOS complexes can lead to insufficient ATP production causing energy deficits in cells and tissues. For example complex I defects can contribute to degenerative disorders linked with lease syndrome through dysregulated energy metabolism. Variations in expression of components within the OXPHOS system such as mutations or deletions can also influence the risk and progression of these diseases.
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Rat liver mitochondria labeled with ab110413 (MS604). The sample in lane 1 was kept at room temperature, whereas the remaining three samples were heated to 37°C, 50°C, and 100°C, respectively. This blot shows that boiling of samples leads to a decrease in signal due to aggregation of proteins therefore heating samples at or close to boiling is not recommended.
Isolated mitochondria from mice brain (control and Alzheimer's disease (AD)) labeled with ab110413 at 1/1000 dilution in 5% BSA.
This image shows NADH dehydrogenase beta subcomplex subunit 8 of Complex I (NDUFB8), succinate dehydrogenase subunit B of Complex II (SDHB), cytochrome c oxidase subunit 1 of Complex IV (MTCO1), cytochrome b-c1 complex subunit 2 of Complex III (UQCRC2) and ATP synthase subunit alpha of Complex V (ATP5A).
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Isolated mitochondria from heart of human (5 μg - Lane 1), cow (1 ug - Lane 2), rat (10 μg - Lane 3), mouse (10 μg - Lane 4) labeling CI subunit NDUFB8 with ab110242 at 0.5 ug/ml.
Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.
Isolated mitochondria from heart of human (5 μg - Lane 1), cow (1 ug - Lane 2), rat (10 μg - Lane 3), mouse (10 μg - Lane 4), HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate (20 μg -Lane 5) labeling CII-30kDa with ab14714 at 5 μg/mL. Secondary antibody is a goat anti-mouse antibody.
Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.
Human skeletal muscle tissue lysate, (10 μg - Lane 1) and Ramos (human Burkitt's lymphoma cell line) whole cell lysate (10 μg - Lane 2) labeling CIII-Core protein 2 with ab14745 at 5 μg/mL. Secondary antibody is a goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP), 1/3000 dilution.
Isolated mitochondria from heart of human (5 μg - Lane 1), cow (1 ug - Lane 2), rat (10 μg - Lane 3), mouse (5 μg - Lane 4) labeling CIV subunit I with ab14705 at 0.5 μg/mL. Secondary antibody is a goat anti-mouse antibody.
Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.
Isolated mitochondria from heart of human (10 μg - Lane 1), cow (4 μg - Lane 2), rat (10 μg - Lane 3), mouse (10 μg - Lane 4), HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate (20 μg -Lane 5) labeling CV alpha subunit with ab14748 at 1 μg/mL
Human liver tissue lysate, (10 μg - Lane 1) and HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate (10 μg - Lane 2) labeling CV alpha subunit with ab14748 at 1 μg/mL. Secondary antibody is a goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP), 1/3000 dilution.
Western Blotting using Total OXPHOS Rodent WB Antibody Cocktail, ab110413. Publication image from Phielix, E. et al., 2017, Nutr Metab (Lond), 28616059. Legend direct from paper.
Effect of postnatal Concept diet on relative protein expression of 5 oxidative phosphorylation complex (OXPHOS) subunits (a – e) in the retroperitoneal (RP) white adipose tissue (n = 8 for CTRL, n = 7 for Concept and n = 10 for REF group) and (f) total protein and western blot bands of one sample per group, samples came from one blot, but not from adjacent lanes, a picture of the whole blot is added as Additional file 1. Early diet and WSD challenge effects were analyzed separately. Difference between Concept and REF group not tested, as groups were fed different postnatal and adult diets. *: p < 0.05, Concept different from CTRL group; †: p 0.05–0.1 REF different from CTRL group.
Western Blotting using Total OXPHOS Rodent WB Antibody Cocktail, ab110413. Publication image from Karch, J. et al., 2017, Elife, 29148970. Legend direct from paper.
Bax and Bak localize to lysosomes and targeting Bax to lysosomes restores autophagic cell death in DKO MEFs.(A) Western blot analysis of the indicated protein fractions from starved and control WT MEFs for Bax, Bak, Lamp1 and the oxidative phosphorylation mitochondrial proteins UQCRC2 (upper band) and MTCO1 (lower band). (B) Transmission EM image of DKO MEFs treated with an adenovirus for mini SOG-Bax. The yellow arrows show electron dense mini-SOG-Bax within the mitochondrial membranes while the pink arrow heads show electron dense regions within the autolysosomes and lysosomes. Scale bar = 1 μm. (C) DKO MEFs infected with a Dox-inducible adenovirus for Bax containing the lysosomal targeting sequence of Lamp1 on its C-terminus. The infected cells were then treated with or without Dox to induce expression and then subjected to serum starvation to induce autophagic cell death, or treated with 200 nM staurosporine to induce apoptotic cell death. Cell death was determined by plasma membrane rupture. All assays are an average or are representative of three independent experiments. The error bars represent the standard error of the mean. *p<0.05 vs no Dox with students t-test.10.7554/eLife.30543.010Figure 4—source data 1.Raw western gel images for Figure 4A.Raw western gel images for Figure 4A.
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