Ndufs4

Manufactured by Abcam

Sourced in United Kingdom

NDUFS4 is a subunit of the mitochondrial respiratory complex I. It is an essential component of the enzyme complex that is responsible for the transfer of electrons in the electron transport chain, which is a key process in cellular respiration and energy production.

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Lab products found in correlation

Ripa buffer, by Merck Group (1 mentions) Oxphos cocktail, by Abcam (1 mentions) Vinculin, by Cell Signaling Technology (1 mentions) Atp5b, by Santa Cruz Biotechnology (1 mentions) Atp5a, by Santa Cruz Biotechnology (1 mentions) Cox 1, by Thermo Fisher Scientific (1 mentions) Atpif1, by Cell Signaling Technology (1 mentions) Histone 3, by Cell Signaling Technology (1 mentions) β actin, by Merck Group (1 mentions) α tubulin, by Merck Group (1 mentions) Hif 1α, by Abcam (1 mentions) Ndufb6, by Abcam (1 mentions) Bsa protein assay, by Thermo Fisher Scientific (1 mentions) Pf 573228, by Selleck Chemicals (1 mentions) Plx4032, by Selleck Chemicals (1 mentions) Ndufa9, by Abcam (1 mentions) Itgb1, by Cell Signaling Technology (1 mentions) Cox5a, by Abcam (1 mentions) Itga5, by Cell Signaling Technology (1 mentions) C myc, by Cell Signaling Technology (1 mentions)

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Anti-NDUFS4 (2 citations)

6 protocols using ndufs4

Muscle Protein Expression Analysis

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For whole-muscle western blot analysis, approximately 50 mg gastrocnemius muscle was ground to a powder using a mortar and pestle in the presence of liquid nitrogen. Thereafter, the powder was resuspended in an amount of RIPA buffer (Sigma), homogenized for 60 s, and then centrifuged at 7,000 × g for 5 min. The supernatant was collected and protein concentration determined using the BradfordUltra method.
Samples were diluted in Laemmli buffer to yield between 20–60 μg of protein per sample that were separated by a 12% SDS-PAGE with proteins subsequently being transferred to nitrocellulose and thereafter probed against Cu/Zn SOD (SOD1) (Enzo), MnSOD (SOD2) (Abcam), NDUFS4 (Abcam), ENO3 (Abcam), and anti-eIF6 (produced in-house).

Clarke K., Ricciardi S., Pearson T., Bharudin I., Davidsen P.K., Bonomo M., Brina D., Scagliola A., Simpson D.M., Beynon R.J., Khanim F., Ankers J., Sarzynski M.A., Ghosh S., Pisconti A., Rozman J., Hrabe de Angelis M., Bunce C., Stewart C., Egginton S., Caddick M., Jackson M., Bouchard C., Biffo S, & Falciani F. (2017). The Role of Eif6 in Skeletal Muscle Homeostasis Revealed by Endurance Training Co-expression Networks. Cell Reports, 21(6), 1507-1520.

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Analyzing Cardiac Protein Profiles

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Heart homogenates or cell lysates were made in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA) with a protease inhibitor cocktail. The protein concentrations were determined using a Pierce BCA Protein Assay Kit. Protein samples (10–20 μg) were loaded per lane on an SDS-polyacrylamide gel. Antibodies were obtained from the following sources: ATPIF1 (catalog 8528), LDHA (catalog 2012), PKM2 (catalog 4053), VDAC (catalog 4661), vinculin (catalog 13901), and histone 3 (catalog 4499) were from Cell Signaling Technology; OXPHOS cocktail (catalog ab110413), cytochrome c core 1 (catalog ab110252), COX-4 (catalog ab14744), and HIF1α (catalog ab1) were from Abcam; NDUFS4 (catalog MA5-19432) and COX-1 (catalog PA5-26688) were from Thermo Fisher Scientific; cytochrome b (catalog sc-11436), ATP5A (catalog sc-136178), and ATP5B (catalog sc-33618) were from Santa Cruz Biotechnology; GFP (catalog 11814460001), β-actin (catalog A2103), and α-tubulin (catalog T6199) were from MilliporeSigma.

Zhou B., Caudal A., Tang X., Chavez J.D., McMillen T.S., Keller A., Villet O., Zhao M., Liu Y., Ritterhoff J., Wang P., Kolwicz SC J.r., Wang W., Bruce J.E, & Tian R. (2022). Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) mediates increased glycolysis in mouse hearts. The Journal of Clinical Investigation, 132(10), e155333.

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Blue Native Gel Electrophoresis of Mitochondrial Complexes

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Complex assembly analysis was carried out by Blue native gel electrophoresis (37 (link)). Sample preparation and Blue native gel electrophoresis were carried out as described previously (38 (link)). Briefly, the cells were suspended in lysis buffer and subjected to homogenization using a Potter-Elvehjem homogenizer with a rotating pestle until 80% of the cells were broken. The homogenate was then centrifuged at 700 g at 4°C to remove nuclear and cytoplasmic debris. The resulting supernatant was again centrifuged at 3000g to pellet the mitochondria. The mitochondrial protein concentration was determined using a BSA protein assay from Thermo-Fisher (Waltham, MA). For Western blotting and immunodetection, antibodies against complex I subunits NDUFA13 (Abcam, Cambridge, GBN), NDUFB6 (Abcam), NDUFS4 (Abcam) and NDUFA9 (Santa Cruz Biochemicals Dallas, TX) were utilized. The Western blots were carried out according to the protocol provided by Mitosciences. In-gel activity assays for Complex I and Complex V were conducted as described before (39 (link)). 2D-SDS electrophoresis was performed as previously described. Lanes from the Blue native gel were cut out and laid onto 10-16% gradient Tricine SDS PAGE gel as published previously (40 (link)).

Vartak R., Deng J., Fang H, & Bai Y. (2015). Redefining the roles of mitochondrial DNA-encoded subunits in respiratory Complex I assembly. Biochimica et biophysica acta, 1852(7), 1531-1539.

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Mitochondrial Complexes Immunohistochemistry

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Immunohistochemical staining of complex I (subunit NDUFS4, Abcam, Cambridge, UK), complex II (subunit SDHA, Mitosciences, Eugene, OR, USA), complex III (subunit core 2, Mitosciences, Eugene, OR, USA), complex IV (subunit I, Mitosciences, Eugene, OR, USA), complex V (subunit alpha, Mitosciences, Eugene, OR, USA), and porin (31HL, Mitosciences, Eugene, OR, USA) were performed as described previously [13 ]. Scale bars in the figures correspond to 100 µm.

Zimmermann F.A., Neureiter D., Sperl W., Mayr J.A, & Kofler B. (2018). Alterations of Oxidative Phosphorylation Complexes in Papillary Thyroid Carcinoma. Cells, 7(5), 40.

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Integrin and Transcription Factor Regulation

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PLX4032, PD98059, AZD6244 and PF-573228 were purchased from Selleck Chemicals. The siRNAs against TCF4 (sc-61657), c-Myc (sc-29226), TCF3 (sc-36618) or TCF12 (sc-35552) were purchased from Santa Cruz Biotechnology. Antibodies against ITGA4, ITGA5, ITGB1, ITGB3, ITGB4, ITGB5, FAK, c-Myc, TCF12, and Porin were purchased from Cell Signaling Technology; p-FAK (Y397) antibodies were from Cell Signaling and Thermo Fisher Scientific, ID2 antibodies were from Cell Signaling, Santa Cruz Biotechnology and Thermo Fisher; V5, HMB45, COX5A, NDUFS4 and NDUFA9 antibodies were from Abcam; TCF4 antibodies were from Abnova and Santa Cruz; and PGC1α antibodies were from Santa Cruz and Millipore.

Luo C., Lim J.H., Lee Y., Granter S.R., Thomas A., Vazquez F., Widlund H.R, & Puigserver P. (2016). A PGC1α-mediated transcriptional axis suppresses melanoma metastasis. Nature, 537(7620), 422-426.

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Quantitative Western Blot Analysis of Protein Modifications

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Western blotting was performed using the Bio-Rad Criterion System. Proteins were resolved on Criterion TGX gels at 200V for 1 hour and transferred to PVDF membranes at 40 mA overnight or 250 mA for 100 minutes. Detection of protein succination was performed as previously described using a polyclonal anti-2SC antibody (Nagai 2007). Antibodies for PPARγ, cleaved caspase 3, GAPDH, eIF2α, p-eIF2α, succinate dehydrogenase (SDHA), fumarase, PDI, calreticulin, and Ero1-Lα were from Cell Signaling Technologies (Danvers, MA). NDUFS4 was from Abcam (Cambridge, MA). β-tubulin, and Grp78 were from Santa Cruz Biotech (Dallas, TX). Monoclonal anti-CHOP was from Thermo Fisher Scientific (Waltham, MA). Anti-adiponectin was from R&D systems (Minneapolis, MN). Chemiluminescent signals were captured on photographic film (Denville Scientific, Metuchen, NJ). Image J software (NIH) was used to quantify band intensity by densitometry.

Tanis R.M., Piroli G.G., Day S.D, & Frizzell N. (2014). The Effect of Glucose Concentration and Sodium Phenylbutyrate Treatment on Mitochondrial Bioenergetics and ER Stress in 3T3-L1 Adipocytes. Biochimica et biophysica acta, 1853(1), 213-221.

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