Ndufb8

Manufactured by Abcam

Sourced in United States, United Kingdom

NDUFB8 is a subunit of the NADH:ubiquinone oxidoreductase (Complex I), the largest enzyme complex of the mitochondrial electron transport chain. It plays a role in the transfer of electrons from NADH to the respiratory chain.

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Lab products found in correlation

Uqcrc2, by Abcam (6 mentions) Ndufa9, by Abcam (5 mentions) Atp5a, by Abcam (5 mentions) Tom20, by Santa Cruz Biotechnology (4 mentions) Chemidoc mp imaging system, by Bio-Rad (4 mentions) β actin, by Merck Group (4 mentions) Gapdh, by Merck Group (2 mentions) Cox5a, by Abcam (2 mentions) Ndufa9, by Thermo Fisher Scientific (2 mentions) P0260, by Agilent Technologies (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Mtco1, by Abcam (2 mentions) Core 2, by Abcam (2 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Quantity one image analysis software, by Bio-Rad (1 mentions) Luminata forte western hrp substrate, by Merck Group (1 mentions) Tomm20, by Abcam (1 mentions) Mgme1, by Merck Group (1 mentions) Endog, by Abcam (1 mentions) Ampk alpha, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Anti-NDUFB8 (3 citations) Mouse anti-NDUFB8 (2 citations)

19 protocols using ndufb8

Quantitative Mitochondrial Protein Analysis

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Total cell lysates were analyzed by SDS PAGE and immunoblotting. Primary antibodies used for western blotting were as follows: ATP5a, UQCR2, SDHB, COII, NDUFB8 (Abcam 110411, 1: 1000); 49KDa (home made, 1: 500); NDUFA9 (Abcam 14713, 1: 1000); H3 (Abcam 10799, 1: 10 000); Twinkle (home made, 1: 1000); TFAM (home made, 1: 5000); exoG (Proteintech 21523-1-AP, 1: 500); MGME1 (Sigma HPA040913, 1: 200); a-tubulin (Sigma T9026, 1: 5000); endoG (Abcam 9647, 1: 1000); Fen1 (home made, 1: 1000); PDH (MitoSciences MSP07, 1: 2500); Tomm20 (Abcam 56783, 1: 500); LC3B (Abcam 48394, 1: 2000); PINK1 (Abcam 75487, 1:1000). Secondary antibodies were as follows: Rabbit (HRP) anti-mouse (Sigma A9044, 1: 10 000) and Goat (HRP) anti-rabbit (Abliance BL2407, 1: 10 000). The detection of proteins was performed using Luminata forte western HRP Substrate (Millipore) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). The relative abundance of specific proteins was evaluated by densitometric quantification of signal intensity, and analyzed using Quantity One image analysis software (BioRad).

Moretton A., Morel F., Macao B., Lachaume P., Ishak L., Lefebvre M., Garreau-Balandier I., Vernet P., Falkenberg M, & Farge G. (2017). Selective mitochondrial DNA degradation following double-strand breaks. PLoS ONE, 12(4), e0176795.

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Antibody Immunodetection Protocol

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The following primary antibodies were used in this study: BrdU (Biorad, MCA2060 or Abcam, ab6326, 1:200 dilution); DNA (Progen, AC-30-10, 1:250 dilution); GAPDH (Sigma, G8795 or Abcam, ab8245, 1:10,000 and 1:2000 dilutions, respectively); GRP78 (Santa Cruz Biotech, Sc-13968, 1:1000 dilution); HSP60 (Abcam, ab46798, 1:1000 dilution); LC3B (Sigma, L7543) 1:5000; MTCO-2 (Abcam, ab110258 1:1000 dilution); NDUFB8 (Abcam, ab110242, 1:1000 dilution); AMPK alpha (Cell Signaling, 2532, 1:1000 dilution); phosphoAMPK alpha (Cell Signaling, 2531, 1:1000 dilution); TOM20 (Santa Cruz Biotech or Abcam, Ab186735, 1:4000 and 1:10,000 dilutions, respectively); VCL (Abcam, ab18058, 1:1000 dilution); and proliferating cell nuclear antigen (PCNA) (Mouse, sc-56, 1:8000 dilution).
Secondary Antibodies: Anti-Mouse IgG (H + L), HRP Conjugate (Promega, W4021, 1:4000 dilution); anti-Rabbit IgG (H + L), HRP Conjugate (Promega, W4011 1:4000 dilution); Alexa Fluor^®−488 goat- anti-mouse (Invitrogen, A-10684,1:1000 dilution); Alexa Fluor^®−568 goat- anti-mouse (Invitrogen, A-11004, 1:1000 dilution); Alexa Fluor^® 568 donkey anti-rabbit (Invitrogen, A-10042, 1:1000 dilution); Alexa Fluor^®-488 goat- anti-rat (Invitrogen, A-11006, 1:1000 dilution).

Pantic B., Ives D., Mennuni M., Perez-Rodriguez D., Fernandez-Pelayo U., Lopez de Arbina A., Muñoz-Oreja M., Villar-Fernandez M., Dang T.M., Vergani L., Johnston I.G., Pitceathly R.D., McFarland R., Hanna M.G., Taylor R.W., Holt I.J, & Spinazzola A. (2021). 2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA. Nature Communications, 12, 6997.

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Brain Region Protein Expression Analysis

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Mice were perfused with phosphate-buffered saline; brains were isolated and different regions were dissected and homogenized in PBS supplemented with complete protease inhibitor mixture (Roche) [13 (link)]. Protein concentrations were determined using the DC kit (Bio-Rad Laboratories). The homogenates were resolved on SDS-PAGE gels, transferred onto a polyvinylidene difluoride (PVDF) membrane, and hybridized with the antibodies raised against Cyt c (Abcam), Cox1 (Mitosciences), NDUFB8, SDHA, UQCRFS1/Rieske, VDAC1/Porin (Abcam); GFAP (Cell Signaling), TUJ1 (Chemicon), Bcl2, BAD, BAX (Cell Signaling); β-actin, γ-tubulin, vinculin (Sigma); SOD1, SOD2, GPX1 (Abgent).
Depending on the size of the related protein, bands were normalized for housekeeping gene (β-actin, γ-tubulin, vinculin) or for total protein loading (visualized by stain free technology, in the Chemidoc system, Biorad). As the same blots were used for hybridization of multiple antibodies, the following figures used the same loading control: hippocampus 4w (Bcl2 and TUJ1; SOD2 and GFAP), hippocampus 8w (Bcl2 and TUJ1; BAX and SOD2; BAD and GFAP), hippocampus 12w (Bcl2 and GFAP), cortex 4w (BAD, SOD2, and GFAP; Bcl2 and TUJ1), cortex 8w (BAX, SOD2, and GFAP; BAD and TUJ1; Bcl2 and GFAP), and cortex 12w (Bcl2 and GFAP).

Pinto M., Vempati U.D., Diaz F., Peralta S, & Moraes C.T. (2018). Ablation of Cytochrome c in Adult Forebrain Neurons Impairs Oxidative Phosphorylation Without Detectable Apoptosis. Molecular neurobiology, 56(5), 3722-3735.

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Mitochondrial Protein Expression Analysis

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The Eu was purchased from National Institutes for Food and Drug Control (China). Dulbecco’s modified Eagle medium (DMEM), Nutrient Mixture F-12 (DMEM/F12) media, horse serum, trypsin and penicillin/streptomycin, were obtained from Life Technologies, GibcoBRL (Rockville,MD,USA). Antibodies against CPT-1C and Tomm20 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Antibodies against ERRα and c-Myc were obtained from Cell Signaling Technology (Beverly, MA,USA) and an antibody against the HRAS, PGC-1β, NDUFB8, SDH-B, UQCRC2, ATP5A, COX5A, MCAD and PPARα were obtained from Abcam (Cambridge, MA, USA). 10058F4 were purchased from Sigma-Aldrich (St.Lois, MO, USA). XF Palmitate-BSA FAO Substrate was obtained from seahorse Bioscience (North Billerica, MA, USA).

Yan X., Zhang G., Bie F., Lv Y., Ma Y., Ma M., Wang Y., Hao X., Yuan N, & Jiang X. (2017). Eugenol inhibits oxidative phosphorylation and fatty acid oxidation via downregulation of c-Myc/PGC-1β/ERRα signaling pathway in MCF10A-ras cells. Scientific Reports, 7, 12920.

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Immunoblotting of Mitochondrial Proteins

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The following primary antibodies were used for immunoblotting: NDUFA9 (Molecular Probes, Eugene, OR, USA, A21344), NDUFB8 (Abcam, Cambridge, UK, ab110242), SDHA (MitoSciences, Eugene, OR, USA, MS204), UQCRC2 (Abcam, ab14745), COX1 (Abcam, ab14705) and COX2 (Molecular Probes, A6404), ATP5A (Abcam, ab14748), ATPB (Abcam, ab14730) and TOM20 (Santa Cruz, Heidelberg, Germany, sc11415). HRP-conjugated anti-mouse or anti-rabbit secondary antibodies were used (P0260 and P0399 respectively; Dako, Glostrup, Denmark). Chemiluminescence ECL Prime Kit (Amersham, Little Chalfont, UK) and ChemiDocMP Imaging System (Bio-Rad, Hemel Hempstead, UK) were used for signal detection and Image lab 4.0.1 (Bio-Rad) software for analysis.

Oláhová M., Haack T.B., Alston C.L., Houghton J.A., He L., Morris A.A., Brown G.K., McFarland R., Chrzanowska-Lightowlers Z.M., Lightowlers R.N., Prokisch H, & Taylor R.W. (2014). A truncating PET100 variant causing fatal infantile lactic acidosis and isolated cytochrome c oxidase deficiency. European Journal of Human Genetics, 23(7), 935-939.

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Mitochondrial and Cell Cycle Markers

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The following antibodies were used: NDUFB8 (Abcam ab110242), SDHA (Cell Signaling Technology 11998), UQCR2 (Abcam ab14745), Tom20 (Santa Cruz Biotechnology sc-11415), p-Rb ser807/811 (Cell Signaling Technology 9308), Cyclin A (Santa Cruz Biotechnology sc-751), p21 (Santa Cruz Biotechnology sc-471 and HUGO 291 Abcam ab107099), IL8 (Abcam ab18672), p-JNK Thr183/Tyr188 (Cell Signaling Technology 9251), JNK (Cell Signaling Technology 9252 and Santa Cruz Biotechnology sc-7345), γH2AX ser139 (Millipore-Merck 05-636 and Cell Signaling Technology 9718), 53BP1 (Abcam 21083 and Cell Signaling Technology 4937), H4K16ac (Millipore-Merck 07-329), H4 (Active Motif 39163), H3K9ac (Active Motif 61252), H3 (Active Motif 39763), cleaved caspase 3 (Cell Signaling Technology 9661), p-38 MAPK Thr180/Tyr182 (Cell Signaling Technology 9211), p38 MAPK (Cell Signaling Technology 9212), p-S6 ser235/236 (Cell Signaling Technology 2211), pS6 (Cell Signaling Technology 2317), IRAK1 (Santa Cruz Biotechnology 5288), Actin (Sigma-Aldrich A1978), GAPDH (Cell Signaling Technology 5174), IL1α (R&D Systems AF-400), and 4-HNE (JaICA).

Vizioli M.G., Liu T., Miller K.N., Robertson N.A., Gilroy K., Lagnado A.B., Perez-Garcia A., Kiourtis C., Dasgupta N., Lei X., Kruger P.J., Nixon C., Clark W., Jurk D., Bird T.G., Passos J.F., Berger S.L., Dou Z, & Adams P.D. (2020). Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence. Genes & Development, 34(5-6), 428-445.

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Cardiac Mitochondrial Protein Profiling

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Mouse cardiac tissue was homogenized in a mixture containing radioimmunoprecipitation assay buffer (Cat: C1053, Beijing Applygen Technologies Inc., China) in the presence of a protease inhibitor cocktail (Cat: G6521, Promega, USA) and the protein content was determined using BCA protein assay kit (Cat: 23227, Pierce, USA). Then protein (20 µg of protein per lane) was subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (Pall Corp., USA) according to standard procedures. The membranes were blocked for 2 h with 5% nonfat milk in TBST (Tris Buffered Saline Tween). Membranes were incubated with each primary antibody (1:500–1:2000 dilution) overnight at 4 °C followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilution). Antibody binding was detected using the SuperSignal West Pico kit (Pierce, USA) according to the manufacturer’s instructions. Antibodies to SDHB (1:1000; Cat: GTX113833), ATP5A1 (1:1000; Cat: GTX101741), UQCRC2 (1:10,000; Cat: GTX114873) were purchased from GeneTex. NDUFB8 (1:5000; Cat: ab192878) was purchased from Abcam. Alpha Tubulin (1:5000; Cat: 66031-1-Ig) was purchased from Proteintech.

Zhao M., Wei H., Li C., Zhan R., Liu C., Gao J., Yi Y., Cui X., Shan W., Ji L., Pan B., Cheng S., Song M., Sun H., Jiang H., Cai J., Garcia-Barrio M.T., Chen Y.E., Meng X., Dong E., Wang D.W, & Zheng L. (2022). Gut microbiota production of trimethyl-5-aminovaleric acid reduces fatty acid oxidation and accelerates cardiac hypertrophy. Nature Communications, 13, 1757.

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Immunoblotting of OXPHOS Complexes

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The following primary antibodies were used for immunoblotting: NDUFA9 (Molecular Probes, A21344), NDUFB8 (Abcam, ab110242), SDHA (MitoSciences, MS204), UQCRC2 (Abcam, ab14745), COX1 (Abcam, ab14705) and COX2 (Molecular probes, A6404), ATP5A (Abcam, ab14748), ATPB (Abcam, ab14730) and TOM20 (Santa Cruz, sc11415). HRP conjugated anti-mouse or anti-rabbit secondary antibodies were used (Dako, P0260 and P0399 respectively). Chemiluminescence ECL Prime Kit (Amersham) and ChemiDocMP Imaging System (Bio-Rad) were used for signal detection and Image lab 4.0.1 software for analysis.

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Western Blotting Analysis of Skeletal Muscle

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Western blotting was performed as previously referenced (37 (link)). Whole gastrocnemius skeletal muscle was pulverized and homogenized in Triton X-100 solution containing protease and phosphatase inhibitors using a tissue homogenizer (TissueLyser LT, Qiagen, Valencia, CA). Total protein value was determined using the Thermo Scientific Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Samples containing the protein homogenates (40 ug of protein) and laemmli buffer were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and were probed with primary antibodies (1:1000 concentration in 5% bovine serum albumin). Oxidative phosphorylation (OX PHOS) complexes antibody containing complex I subunit NDUFB8, complex II-30kDa, complex IV subunit I, and complex V alpha subunit was purchased from Abcam (ab110413, Abcam, Cambridge, MA), and total adenosine monophosphate-activated protein kinase (AMPK)-α, pAMPK Thr¹⁷² (activation site) and (B) pAMPK Ser^485/491 (inhibition site) antibodies from Cell Signaling (#2532, #2531, and #4185, respectively; Beverly, MA). Individual protein bands were quantified using a densitometer (Bio-Rad), and normalized to β-Actin antibody (#4967, Abcam, Cambridge, MA).

Park Y.M., Kanaley J.A., Zidon T.M., Welly R.J., Scroggins R.J., Britton S.L., Koch L.G., Thyfault J.P., Booth F.W., Padilla J, & Vieira-Potter V.J. (2016). Ovariectomized High Fit Rats Are Protected against Diet-Induced Insulin Resistance. Medicine and science in sports and exercise, 48(7), 1259-1269.

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Detailed Western Blot Antibodies

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Western blots were performed and developed as described in Ref. [39] using antibodies as follows: 6XHis, VDAC, uL3m, mS29, SLIRP, COX2, NDUFA9, NDUFB8 (Abcam, Cambridge, UK); AIF, uL11m (New England Biolabs); eIF4E (Cell Signaling Technology, Danvers, MA, USA); SDHA (Thermo Fisher Scientific); mL45, mS40, mS22 (ProteinTech Group, Rosemont, IL, USA); LRPPRC (Santa Cruz Biotechnology, Dallas, TX, USA); β‐actin (Sigma‐Aldrich, St. Louis, MO, USA); and GDH, EFTu (custom made).

Bruni F., Proctor‐Kent Y., Lightowlers R.N, & Chrzanowska‐Lightowlers Z.M. (2020). Messenger RNA delivery to mitoribosomes – hints from a bacterial toxin. The Febs Journal, 288(2), 437-451.

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