Ab14711

Manufactured by Abcam

Ab14711 is a primary antibody that recognizes the human GAPDH protein. It is suitable for use in various immunoassay applications such as Western blotting, immunohistochemistry, and ELISA.

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Lab products found in correlation

Ab14748, by Abcam (7 mentions) Ab14715, by Abcam (3 mentions) Ab14705, by Abcam (3 mentions) Ab14745, by Abcam (2 mentions) Ab14744, by Abcam (2 mentions) Ab14714, by Abcam (2 mentions) A21351, by Thermo Fisher Scientific (2 mentions) Ab33985, by Abcam (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Ab6556, by Abcam (2 mentions) Ab110259, by Abcam (1 mentions) Ab87399, by Abcam (1 mentions) A1978, by Merck Group (1 mentions) Ab7291, by Abcam (1 mentions) P0260, by Agilent Technologies (1 mentions) P0399, by Agilent Technologies (1 mentions) Ab24733, by Abcam (1 mentions) Ab110242, by Abcam (1 mentions) Ab110258, by Abcam (1 mentions) Immun star hrp luminol enhancer, by Bio-Rad (1 mentions)

15 protocols using ab14711

Mitochondrial Protein Complex Analysis

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Mitochondrial pellets were solubilized with digitonin at 4:1 g protein and fractionated in blue native (BN) gels [28 (link)]. A total of 30 μg of mitochondrial proteins were fractionated in NativePAGE 3–12% Bis-Tris Protein Gels, 1.0 mm, 10-well (Invitrogen) at 4°C using cathode (50 mM Tricine, 15 mM Bis-Tris, pH 7.0 and 0.02% Serva blue G) and anode buffers (50 mM Bis-Tris, pH 7.0). Electrophoresis was carried out at a constant voltage (70 V) until the samples entered the polyacrylamide gradient (approximately 30 minutes) and then at a constant current (15 mA) until the dye reached the end of the gel (approximately 1 hour). After fractionation, the gels were electroblotted onto PVDF membranes and processed for immunoblot analysis as described above. The following primary antibodies were used: mouse anti-NADHs9 (NDUFA9, clone 15/22-5 [89 (link)], 1:1,000), mouse anti-NDUFS3 (clone 17D95, Abcam, ab14711, 1:1,000), mouse anti-NDUFS4 (clone 2C7CD4AG3, Abcam, ab87399, 1:1,000), mouse anti-SDH-A (clone 2E3GC12FB2AE2, Abcam, ab14715, 1:1,000), mouse anti-UQCRC2 (clone 13G12AF12BB11, Abcam, ab14745, 1:1,000), mouse anti-MT-CO1 (clone 1D6E1A8, Invitrogen, #459600, 1:1,000), mouse anti-MT-CO3 (clone DA5BC4, Abcam, ab110259, 1:1,000), mouse anti-COX IV (clone 20E8C12, Abcam, ab14744, 1:1,000), and rabbit anti-β-F1 [93 (link)] (1:20,000).

Esparza-Moltó P.B., Romero-Carramiñana I., Núñez de Arenas C., Pereira M.P., Blanco N., Pardo B., Bates G.R., Sánchez-Castillo C., Artuch R., Murphy M.P., Esteban J.A, & Cuezva J.M. (2021). Generation of mitochondrial reactive oxygen species is controlled by ATPase inhibitory factor 1 and regulates cognition. PLoS Biology, 19(5), e3001252.

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Western Blot Analysis of Skeletal Muscle

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Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.³⁰ (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.

Feichtinger R.G., Oláhová M., Kishita Y., Garone C., Kremer L.S., Yagi M., Uchiumi T., Jourdain A.A., Thompson K., D’Souza A.R., Kopajtich R., Alston C.L., Koch J., Sperl W., Mastantuono E., Strom T.M., Wortmann S.B., Meitinger T., Pierre G., Chinnery P.F., Chrzanowska-Lightowlers Z.M., Lightowlers R.N., DiMauro S., Calvo S.E., Mootha V.K., Moggio M., Sciacco M., Comi G.P., Ronchi D., Murayama K., Ohtake A., Rebelo-Guiomar P., Kohda M., Kang D., Mayr J.A., Taylor R.W., Okazaki Y., Minczuk M, & Prokisch H. (2017). Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies. American Journal of Human Genetics, 101(4), 525-538.

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Mitochondrial Isolation and Protein Analysis

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Isolation of mitochondria from mouse heart and colon was performed by differential centrifugation (46 (link)). Twenty micrograms of isolated mitochondria or 50 μg of total protein extracts was resuspended in 4× Laemmli buffer. Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) using 12% precast gels (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (GE Healthcare). Immunodetection was performed according to standard techniques using enhanced chemiluminescence Immun-Star HRP Luminol/Enhancer (Bio-Rad). The following antibodies were used: SDHA (ab14715, Abcam), Ndufs3 (ab14711, Abcam), VDAC/Porin (ab128568, Abcam), ATP synthase subunit alpha (Molecular Probes A21350, Thermo Fisher Scientific), UQCRC1 CIII subunit core1 (Molecular Probes A21362, Thermo Fisher Scientific), COX I (459600, Invitrogen), TFAM (rabbit polyclonal antisera against TFAM generated using recombinant mouse protein), and GAPDH (ab8245, Abcam).

Filograna R., Koolmeister C., Upadhyay M., Pajak A., Clemente P., Wibom R., Simard M.L., Wredenberg A., Freyer C., Stewart J.B, & Larsson N.G. (2019). Modulation of mtDNA copy number ameliorates the pathological consequences of a heteroplasmic mtDNA mutation in the mouse. Science Advances, 5(4), eaav9824.

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Drosophila Proteasomal Subunit Antibodies

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The antibody against the Drosophila Prosβ5 proteasomal subunit was a kind gift of Maria Figueiredo-Pereira (Hunter College, New York, USA). Primary antibodies against the 20S-α (sc-65755) and Rpn7 (sc-65750) Drosophila proteasome subunits; anti-Ubiquitin (Ub) (sc-8017), anti-β-Tubulin (sc-20852), anti-Hsp70 (sc-25837), anti-GFP (sc-9996) and the HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology. The antibody against Gapdh (G9545) was from Sigma-Aldrich and the anti-Rabbit-IgG Alexa Flour 647 conjugated antibody (711-605-152) was from Jackson ImmunoResearch. The antibody against the Histone H2AvD (600-401-914) was obtained from Rockland Inc. The antibody against ref(2) has been described before [84]. The antibodies against NDUFS3 (ab14711), ATP5PF/complex V subunit-ATP5F1A (ab14748) and HSPA9/Grp75 (ab2799) were from Abcam and the antibody against PSMD11 (Rpn6, NBP1-46191) was from Novus.

Tsakiri E.N., Gumeni S., Vougas K., Pendin D., Papassideri I., Daga A., Gorgoulis V., Juhász G., Scorrano L, & Trougakos I.P. (2019). Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy. Autophagy, 15(10), 1757-1773.

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Mitochondrial protein complex analysis

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Mitochondrial protein complex samples for BN-PAGE were prepared from cultured primary cortical astrocytes as previously described (Nijtmans et al., 2002 (link)). Digitonin (2 mg/ml)-treated cell pellets were solubilized in 1.5 m aminocaproic acid, 50 mm Bis-Tris-HCl, pH 7.0, and 1% dodecylmaltoside. The samples were incubated on ice for 15 min and centrifuged at 20,000 × g for 20 min to remove insolubilized material. Supernatants containing the mitochondrial protein complexes were collected. BN–PAGE electrophoresis and blotting were performed as previously described (Ugalde et al., 2004 (link)). Briefly, 20-μg samples were combined with 5% Serva blue G and separated on 5–15% gradient acrylamide gel. The proteins were transferred to a nitrocellulose membrane by semi-dry protein transfer. Western blotting was performed using antibodies against NDUFS3 (Abcam, ab14711), ATP5A (Abcam, ab14748), UQCRC2 (Abcam, ab14745), COX I (Molecular probes, A6403), and SDHA (Abcam, ab14715).

Byts N., Sharma S., Laurila J., Paudel P., Miinalainen I., Ronkainen V.P., Hinttala R., Törnquist K., Koivunen P, & Myllyharju J. (2021). Transmembrane Prolyl 4-Hydroxylase is a Novel Regulator of Calcium Signaling in Astrocytes. eNeuro, 8(1), ENEURO.0253-20.2020.

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Immunoblotting of Mitochondrial Proteins

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Immunoblotting was performed as previously described (17 (link)). In addition to the new rabbit polyclonal antibodies we generated, the following primary antibodies were also used: anti-dNDUFS2 (17 (link)), anti-NDUFS3 (Abcam, ab14711), anti-dNDUFS5 (17 (link)), anti-dNDUFS7 (54 (link)), anti-dNDUFS8 (54 (link)), anti-dNDUFA8 (54 (link)), anti-dNDUFA11 (54 (link)), anti-dNDUFA12 (54 (link)), anti-dNDUFV1 (17 (link)), anti-dNDUFV3 (54 (link)), anti-dNDUFB5 (17 (link)), anti-dNDUFB6 (17 (link)), anti-dNDUFB8 (17 (link)), anti-dND1 (17 (link)), anti-dND2 (17 (link)), anti-dND3 (17 (link)), anti-dND4L (17 (link)), anti-dND5 (17 (link)), anti-dND6 (17 (link)), anti-VDAC (Abcam, ab14734), anti-HSP60 (CST, #4870S), anti–cytochrome c (ab13575), anti–phospho-AMPKα (Thr¹⁷²) [Cell Signaling Technology (CST), #2535], anti-HSP90 (CST, #4874), anti–phospho-eIF2α (Ser⁵¹) (CST, #3398), anti–phospho-p44/42 MAPK (Erk1/2) (Thr²⁰²/Tyr²⁰⁴) (CST, #4377), anti–phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) (CST, #9215), anti–phospho-SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵) (CST, #4668), anti–phospho-SEK1/MKK4 (Ser²⁵⁷/Thr²⁶¹) (CST, #9156), and anti-ATPsynβ (Life Technologies, A21351). Secondary antibodies used were goat anti-rabbit horseradish peroxidase (PI31460 from Pierce) and goat anti-mouse horseradish peroxidase (PI31430 from Pierce).

Murari A., Goparaju N.S., Rhooms S.K., Hossain K.F., Liang F.G., Garcia C.J., Osei C., Liu T., Li H., Kitsis R.N., Patel R, & Owusu-Ansah E. (2022). IDH2-mediated regulation of the biogenesis of the oxidative phosphorylation system. Science Advances, 8(19), eabl8716.

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Crista Membrane Purification for Sub-Tomogram Averaging

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Crista membranes used for the sub-tomogram averaging experiments were generated by successive freeze–thaw cycles of mitochondria at −80°C. To purify mitochondrial membranes from other cellular material, membrane extracts were incubated for 1 h at 4°C with an anti-NDUFS3 primary antibody (ab14711; Abcam) against the matrix arm of complex I from C. elegans, followed by a 3 h incubation with an anti-mouse secondary conjugated to a quantum dot emitting at 625 nm (Q22085; Invitrogen). Crista membranes were separated from unbound antibodies and other cellular material on an Optiprep gradient with 10 layers (200 µl volume each) ranging from 0 to 27% v/v of iodixanol in STEG/M buffer, by centrifugation at 80 000×g for 30 min at 4°C using a TLS-55 rotor (Beckman Coulter Inc., Miami, FL, U.S.A.). Crista membranes were identified and removed based on fluorescence under a UV lamp. Samples were then diluted in STEG/M buffer to wash out the iodixanol, and spun at 20 000×g for 15 min at 4°C to pellet the membranes. The enriched cristae were again re-suspended in the STEG/M buffer.

Buzzard E., McLaren M., Bragoszewski P., Brancaccio A., Ford H.C., Daum B., Kuwabara P., Collinson I, & Gold V.A. (2024). The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography. Biochemical Journal, 481(3), 161-175.

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Western Blot Analysis of Mitochondrial Proteins

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Sample preparation (20 flies were used per sample, three biological replicates per group) and Western blotting were performed as described in Scialò et al (2016). The primary antibodies employed together with the appropriate secondary antibodies were as follows: anti‐NDUFS3 (Abcam; ab14711) 1:1,000 diluted in 5% milk in PBS‐Tween 1x, anti‐SDHA (Abcam; ab209986) 1:500 diluted in 5% BSA in PBS‐Tween 1x; anti‐ATP5α (Abcam; ab14748) 1:100,000 diluted in 5% milk in PBS‐Tween 1x, anti‐UQCRC1 (Abcam; ab110252) 1:500 diluted in 5% BSA in PBS‐Tween 1x, anti‐MTCO1 (Abcam; ab14705) 1:500 diluted in 5% BSA in PBS‐Tween 1x, Porin (Abcam; ab14734) 1:1,000 diluted in 5% milk in PBS‐Tween 1x, anti‐beta‐tubulin [EPR16774] (Abcam; ab179513) 1:1,000 diluted in 5% milk in PBS‐Tween 1x, HRP Horse Anti‐Mouse IgG Antibody (Peroxidase; Vector laboratories, California; PI‐2000) 1:5,000 diluted in 5% milk, HRP Goat Anti‐Rabbit IgG Antibody (Peroxidase; Vector laboratories, California; PI‐1000) 1:5,000 diluted in 5% milk. Image quantification: ImageJ was used to quantify the protein band intensities. For each image, the bands were quantified and normalised by the corresponding tubulin intensity used as a loading control.

Thompson K., Mai N., Oláhová M., Scialó F., Formosa L.E., Stroud D.A., Garrett M., Lax N.Z., Robertson F.M., Jou C., Nascimento A., Ortez C., Jimenez‐Mallebrera C., Hardy S.A., He L., Brown G.K., Marttinen P., McFarland R., Sanz A., Battersby B.J., Bonnen P.E., Ryan M.T., Chrzanowska‐Lightowlers Z.M., Lightowlers R.N, & Taylor R.W. (2018). OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect. EMBO Molecular Medicine, 10(11), e9060.

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Fly Protein Extraction and Western Blotting

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For each biological replicate, 10 male and 10 female flies were used. Flies were first homogenised in 40μl 1X sample buffer using a pestle in a 1.5ml Eppendorf and spun down at 14000g for 5 minutes. The 1X sample buffer consisted of 50mM Tris-HCl pH 6.8, 10% v/v glycerol, 2% w/v SDS and 0.01% w/v bromophenol blue. 100mM dithiothreitol (DTT) was added and samples were boiled at 95°C for 5 minutes. Samples were stored at -20°C. Antibodies used were rabbit anti-VDAC (1:1000; ab14374, Abcam), mouse anti-Ndufs3 (1:500; ab14711, Abcam), rabbit anti-actin (1:5000; 4967, Cell Signalling Technology), anti-rabbit phospho-AMPK (Thr172; #2535, Cell Signalling Technology). Total protein staining was performed using the REVERT Total Protein Stain Kit (926–11010, LiCor).

Granat L., Knorr D.Y., Ranson D.C., Hamer E.L., Chakrabarty R.P., Mattedi F., Fort-Aznar L., Hirth F., Sweeney S.T., Vagnoni A., Chandel N.S, & Bateman J.M. (2023). Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency. PLOS Genetics, 19(7), e1010793.

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Mitochondrial Protein Quantification Protocol

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Whole flies were homogenized in RIPA buffer and the supernatant was subjected to SDS-PAGE electrophoresis. Following electrophoresis, proteins were transferred to PVDF membrane, and the membrane was blocked with 5% milk, then incubated overnight with antisera. Antibodies were used at the following concentrations for immunoblots: COX IV (ab33985, Abcam), 1:5000; NDUFS3 (ab14711, Abcam), 1:1000; SDHB (ab14714, Abcam), 1:1000; phospho-EIF2α (ab32157, Abcam), 1:1000; EIF2α (ab26197, Abcam), 1:1000; Lon (NBP1-81734, Novus Biologicals), 1:5000; Hsp60 (4870S, Cell Signaling Technology), 1:1000; mtHsp70 (sc-13967, Santa Cruz Biotechnology), 1:5000; ATP Synthase β (A21351, Thermo Fisher Scientific), 1:1000; and Actin (MAB1501, Chemicon), 1:10,000. Chemiluminescence was used for antibody detection and western blot images were quantified using ImageJ software and normalized to Actin. Each experiment was performed at least three times.

Pareek G., Thomas R.E, & Pallanck L.J. (2018). Loss of the Drosophilam-AAA mitochondrial protease paraplegin results in mitochondrial dysfunction, shortened lifespan, and neuronal and muscular degeneration. Cell Death & Disease, 9(3), 304.

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