Ab110246

Manufactured by Abcam

Sourced in Japan, United States

Ab110246 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but no further details on its core function can be provided in an unbiased and factual manner without risk of extrapolation.

Automatically generated - may contain errors

Lab products found in correlation

Ab14715, by Abcam (4 mentions) Ab14713, by Abcam (4 mentions) Ab79826, by Abcam (2 mentions) Ab106699, by Proteintech (2 mentions) Ab28172, by Abcam (2 mentions) Ab14745, by Cell Signaling Technology (2 mentions) Ab110252, by Abcam (2 mentions) Ab14705, by Abcam (2 mentions) Ab14714, by Abcam (2 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Anti vinculin, by Merck Group (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Protease and phosphatase inhibitor cocktail, by Roche (1 mentions) Ab75973, by Abcam (1 mentions) Ab110413, by Abcam (1 mentions) Dc assay kit, by Bio-Rad (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions) Nbp1 21502, by Novus Biologicals (1 mentions) Ab14745, by Abcam (1 mentions)

7 protocols using ab110246

Mitochondrial Protein Analysis by SDS-PAGE and BN-PAGE

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For SDS PAGE experiments, 20μg of mitochondrial proteins or 15μg of whole cell extracts were loaded in 15% SDS gels. Membranes were probed with the following antibodies: NDUFA9 (1:1000, abcam, ab14713), NDUFAF1 (1:10000, abcam, ab79826), NDUFS3 (1:1000, abcam, ab110246), NDUFB9 (1:1000, abcam, ab106699), NDUFV2 (1:1000, proteintech, 15301-1-AP), Prohibitin (1:1000, abcam, ab28172), SDHA (1:1000, abcam, ab14715), UQCRC2 (1:1000, abcam, ab14745), Tubulin (1:1000, Cell Signaling, #21485). Proteins separated by BN PAGE were transferred to PVDF membranes and western blotting was performed as above. Alternatively, the BN gels were used for in-gel activity assays for complex I by incubating the gel in the assay buffer consisting of 5mM Tris/HCl with 2.5mg/ml nitrotetrazolium blue and 0.1mg/ml NADH (pH 7.4).

Miwa S., Jow H., Baty K., Johnson A., Czapiewski R., Saretzki G., Treumann A, & von Zglinicki T. (2014). Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice. Nature Communications, 5, 3837.

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Protein Extraction and Immunoblotting Protocol

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For protein extract collection, 1.5x10⁶ cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (both from Roche) and subsequently sonicated and boiled for 5 min at 95°C. Protein lysates were separated on 4-12% SDS-gradient gels (Bio-Rad). Proteins were transferred to PVDF membranes (Millipore). Membranes were blocked with 5% bovine serum albumin (BSA) in PBS and probed with antibodies sourced as follows: anti-CORE1 (1/5000 dilution, ab110252, Abcam), -NDUSF3 (1/5000 dilution, ab110246, Abcam), -ATP-B (1/2000 dilution, Abcam), and -NDUFA9 (1/5000 dilution, ab14713, Abcam), -FpSDH (1/5000 dilution, #459200, Invitrogen), -Cox5b (1/5000 dilution, Proteintech Europe), anti-vinculin (1/10000 dilution, Sigma); and anti-β-actin (1/10000 dilution, Santa Cruz Biotechnology).

Garaude J., Acín-Pérez R., Martínez-Cano S., Enamorado M., Ugolini M., Nistal-Villán E., Hervás-Stubbs S., Pelegrín P., Sander L.E., Enríquez J.A, & Sancho D. (2016). Mitochondrial respiratory chain adaptations in macrophages contribute to antibacterial host defence. Nature immunology, 17(9), 1037-1045.

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Muscle Iron Regulation Protein Analysis

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The adductor muscles were homogenized using freeze crusher (SK-100, Tokken Inc., Chiba, Japan) and cell lysis buffer (#9803, Cell Signaling Technology, MA, USA). The protein concentration was determined by DC assay kit (#5000112, Bio-Rad, CA, USA). Thirty micrograms of protein homogenate was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The expression levels of proteins were detected by an enhanced chemiluminescence kit (#32106, Thermo Scientific, IL, USA). Anti-ferritin (#ab75973, Abcam, UK; dilution 1:1000), anti-TfR1 (#13-6800, Invitrogen, CA, USA; dilution 1:1000), anti-ferroportin (NBP1-21502, Novus Biologicals, CO, USA; dilution 1:1000), anti-tubulin (#PM054-7, MBL, Aichi, Japan; dilution 1:1000), anti-mitochondrial total oxidative phosphorylation (OXPHOS) antibody cocktail including antibodies against C I, C II, C III, and C V (#ab110413, Abcam; dilution 1:1000), anti-NADH dehydrogenase iron-sulfur protein 3 (NDUFS3, #ab110246, Abcam; dilution 1:1000), and anti-GAPDH (#2118, Cell Signaling Technology; dilution 1:1000) antibodies were used.

Okuno K., Naito Y., Yasumura S., Sawada H., Asakura M., Masuyama T, & Ishihara M. (2019). Haploinsufficiency of Transferrin Receptor 1 Impairs Angiogenesis with Reduced Mitochondrial Complex I in Mice with Limb Ischemia. Scientific Reports, 9, 13658.

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Mitochondrial Protein Analysis by SDS-PAGE and BN-PAGE

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For SDS–PAGE experiments, 20 μg of mitochondrial
proteins or 15 μg of whole-cell extracts were loaded in 15%
SDS gels. Membranes were probed with the following antibodies: NDUFA9 (1:1,000, Abcam, ab14713),
NDUFAF1 (1:10,000, Abcam,
ab79826), NDUFS3 (1:1,000,
Abcam, ab110246), NDUFB9
(1:1,000, Abcam, ab106699), NDUFV2 (1:1,000, Proteintech, 15301-1-AP), PhB (1:1,000, Abcam, ab28172),
SDHA (1:1,000, Abcam,
ab14715), UQCRC2 (1:1,000,
Abcam, ab14745) and tubulin (1:1,000, Cell Signaling, no. 21485). Proteins
separated by BN PAGE were transferred to Polyvinylidene fluoride (PVDF)
membranes and western blotting was performed as above. Alternatively, the BN
gels were used for in-gel activity assays for complex I by incubating the gel in
the assay buffer consisting of 5 mM Tris/HCl with
2.5 mg ml⁻¹nitrotetrazolium blue and
0.1 mg ml⁻¹NADH (pH 7.4).

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Protein Expression Analysis in Fibroblasts

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Protein gel electrophoresis and blotting analyses were performed on whole cell protein extracts obtained from patient (P) and control (C1 and C2) fibroblasts. Samples containing 30 μg protein were separated by denaturing NuPAGE 4%–12% Bis-Tris gels and transferred to nitrocellulose membrane. Immunodetection was carried out using primary antibodies against target proteins: RNase H1 (ab56560, Abcam), POLG (sc-5931, Santa Cruz), POLG2 (LS-C334882, LSBio), TWNK (gift from M Falkenberg), SSBP1 (ab74710, Abcam), TFAM (gift from RJ Wiesner), POLRMT (ab32954, Abcam), LRPPRC (ab97505, Abcam), SLIRP (ab51523, Abcam), ATAD3 (gift from JE Walker), bL12 (14795-1-AP, Proteintech), uL11 (SAB2701374, Sigma), MDDX28 (ab70821, Abcam), mS35 (16457-1-AP, Proteintech), mS18b (16139-1-AP, Proteintech), NDUFS3 (ab110246, Abcam), NDUFB8 (ab110242, Abcam), SDHA (ab14715, Abcam), SDHB (ab14714, Abcam), UQCRC1 (ab96333, Abcam), UQCRC2 (ab14745, Abcam), MT-CO1 (ab14705, Abcam), MT-CO2 (ab91317, Abcam), COX4l1 (ab14744, Abcam), ATPF1 (ab84625, Abcam), and ATPA1 (ab110273, Abcam), along with GAPDH (ab8245, Abcam), used as loading control. For quantifications, images were digitalized and analyzed with ImageJ software, and data analyses were performed in Microsoft Excel.

Reyes A., Rusecka J., Tońska K, & Zeviani M. (2020). RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA. Frontiers in Genetics, 10, 1393.

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Quantitative Analysis of OXPHOS Proteins

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Proteins were transferred from the gels to PVDF membranes (Immobilon-P, Millipore) using semidry electroblotting. The membranes were blocked with 5% (w/v) non-fat dried milk in TBS (150 mM NaCl, 10 mM Tris, pH 7.5) for 1 h and incubated 2 h or overnight at 4 °C with primary antibodies diluted in TBS with 0.1% Tween-20. Monoclonal primary antibodies to the following enzymes of OXPHOS were used: SDHA (ab14715, Abcam), CORE1 (ab110252, Abcam), NDUFB6 (ab110244, Abcam), NDUFS3 (ab110246, Abcam), COX1 (ab14705, Abcam). The detection of the signals was performed with the secondary Alexa Fluor 680-labeled antibody (Life Technologies) using the Odyssey fluorescence scanner (LI-COR). Quantification of detected signals from BNE/SDS PAGE was carried out in Aida Image Analyzer program, version 3.21.

Kovářová N., Pecina P., Nůsková H., Vrbacký M., Zeviani M., Mráček T., Viscomi C, & Houštěk J. (2016). Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects. Biochimica et Biophysica Acta, 1862(4), 705-715.

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Cardiac Protein Fractionation and Analysis

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Total protein lysates were prepared from rat heart tissues using protein extraction reagent (USA, Thermo, 78510) as described previously.²¹, ²³ A mitochondria/cytosol fractionation kit (UK, Abcam, ab65320) was used for isolation of mitochondrial and cytosolic fractions from cardiac tissues, following the instructions in the manual.²⁴ Protein extracts were first separated through SDS‐PAGE and then transferred to nitrocellulose membranes. The membranes were incubated at 4℃ overnight with appropriate primary antibodies (ISCA1 [USA, Thermo, PA5‐60121]; TOMM20 [USA, Thermo, PA5‐52843]; NDUFA9 [UK, Abcam, ab14713]; NDUFS3 [UK, Abcam, ab110246]; SDHB [UK, Abcam, ab14714]; GAPDH [UK, Abcam, ab201822]) and subsequently kept for 1 h at RT with the appropriate secondary antibodies. Western blot images were acquired (Bio‐Rad, ChemiDoc XRS+Gel Imaging System, USA) and quantitatively analyzed with Image J software.

Ling Y., Ma J., Qi X., Zhang X., Kong Q., Guan F., Dong W., Chen W., Gao S., Gao X., Pan S., Ma Y., Lu D, & Zhang L. (2021). Novel rat model of multiple mitochondrial dysfunction syndromes (MMDS) complicated with cardiomyopathy. Animal Models and Experimental Medicine, 4(4), 381-390.

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