Mitochondria were isolated using Dounce or Balch homogenizers, followed by standard differential centrifugation13 (link),50 (link),70 (link). Experimental procedure and antibodies for NBGE were used as described previously57 (link),71 (link). In brief, digitonin-solubilised mitochondria were separated on NativePAGE Novex Bis-Tris 3–12% gradient gels. After electrophoresis, the gels were incubated in transfer buffer containing 0.1% SDS for 10 min and proteins were transferred to PVDF membranes probed with specific antibodies (all diluted 1:500, except for VDAC1 and HSP60 diluted 1:1000) against complex I (NDUFA9, ab14713, Abcam; or NDUFB8, ab110242, Abcam), CII (SDHA, 14715, Abcam), CIII (Core2, ab14745, Abcam), CIV (COXVa, ab110262, Abcam) and CV (ATP5A, ab14748, Abcam; or ATP5B, HPA001520, Sigma Aldrich), and VDAC1 (ab15895, Abcam) or HSP60 (12165S, Cell Signaling) as the loading control.
Ab110262
Manufactured by Abcam
Sourced in China
Ab110262 is a recombinant protein that serves as a reference standard. It is used for the detection and quantification of target proteins in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Ab14713, by Abcam (4 mentions)
Ab14745, by Abcam (3 mentions)
Ab14748, by Abcam (3 mentions)
Ab15895, by Abcam (2 mentions)
Ab110242, by Abcam (2 mentions)
Hpa001520, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab14715, by Abcam (2 mentions)
Nativepage novex 3 12 bis tris protein gels, by Thermo Fisher Scientific (1 mentions)
Ab14734, by Abcam (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Ab14705, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Amersham ecl prime reagent, by GE Healthcare (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
A3854, by Merck Group (1 mentions)
Ab128743, by Abcam (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
8 protocols using ab110262
1
Isolation and Analysis of Mitochondrial Complexes
2
Mitochondrial OXPHOS Complex Analysis
3
Extraction and Analysis of Mitochondrial Proteins
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To prepare native mitochondrial proteins, pellets were solubilized in 30–100 μL buffer containing 1.5 M aminocaproic acid, 50 mM Bis-Tris, pH 7.0. Mitochondria were extracted with a digitonin:protein ratio of 4g/g. Solubilized samples were incubated on ice for 20 min and centrifuged for 30 min at 13,000 rpm at 4 °C . Then the supernatant was suspended in 750 mM aminocaproic acid, 50 mM Bis-Tris, 0.5 mM EDTA and 5% Serva Blue G-250 prior to loading. Thirty μg of protein were loaded in Native PAGE™ Novex® 3–12% Bis-Tris Protein Gels (Invitrogen). Duplicate gels were used for second-dimension (2D) 10% SDS-PAGE gels. Proteins were transferred at 1.3A constant for 10 min and probed with 1:1000 mouse anti-COX5A (ab110262), 1:1000 mouse anti-NDUFA9 (ab14713) and 1:2000 mouse anti-VDAC1 (ab14734) (Abcam). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibodies. The immunoreactive bands were visualized with Cheluminate-HRP PicoDetect Kit for Western Blotting (Panreac AppliChem).
4
Western Blot Analysis of Mouse Liver Proteins
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Total tissue proteins were obtained by homogenizing mouse liver tissue in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton x-100, 1% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA) including a protease inhibitor cocktail (Sigma, St. Louis, MO) and resolved on SDS-PAGE (Invitrogen, Beijing, China) before transferred to a PVDF membrane and blocked with 5% BSA in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h. The membranes were incubated with primary antibody overnight at 4°C followed by washed three times with TBST then probed with appropriate secondary antibodies for 1 h at room temperature, washed as above and proteins bands were detected using ECL (EMD Millipore, Shanghai, China). Anti-LRPPRC (ab80881, Abcam, Shanghai, China), COX1 (ab14705), COX5A (ab110262), and β-Actin (ab8227) antibodies were diluted according to the manufacturer instructions.
5
Antibody-Based Protein Detection Protocol
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After electroblotting the proteins onto PVDF membranes, they were detected by incubating the membranes with the following antibodies: anti-ATP5A (ab14748, Abcam), anti-ATP5B (ab128743, Abcam), anti-β-actin (A3854, Sigma-Aldrich), anti-COX5A (ab110262, Abcam), anti-NDUFA9 (ab14713, Abcam), anti-NDUFS4 (PA5-92940, Thermo Fisher Scientific), anti-NDUFV1 (sc-100566, Santa Cruz Biotechnology), anti-SDHA (ab14715, Abcam), and anti-UQCR2 (ab14745, Abcam). Immunoreactive bands were detected with an Amersham ECL Prime Reagent (GE- Healthcare) in a ChemiDoc MP Imager (Bio-Rad).
6
Quantitative Muscle Fiber Characterization
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Primary antibodies: Myh2 (3 μg/ml, Developmental Studies Hybridoma Bank, sc-71, AB_2147165), Myh4 (3 μg/ml, Developmental Studies Hybridoma Bank, BF-F3, AB_2266724), Myh7 (3 μg/ml, Developmental Studies Hybridoma Bank, BA-F8, AB_10572253), Desmin (1:20. Thermo Fisher Scientific, MA5-13259), Cox5a (1:1000, Abcam, ab110262, AB_10861723), Glut1 (1:200, Abcam, ab652, AB_305540), Myf5 (1:10,000, Abcam, ab125078, AB_10975611), Fasn (1:1000, Abcam, ab22759, AB_732316). Secondary antibodies: goat-anti-mouse IgG2b Alexa 350 (1:200, Thermo Fisher A-21140, AB_2535777), goat-anti-mouse IgG1 Alexa 488 (1:200, Thermo Fisher A-21121, AB_2535764), goat-anti-mouse IgM Alexa 555 (1:200, Thermo Fisher A-21426, AB_2535847), IRDye 800CW goat anti-mouse IgG Secondary Antibody (1:10,000, Licor, 926-32210, AB_621842), IRDye 800CW goat anti-rabbit IgG Secondary Antibody (1:10,000, Licor, 926-32211, AB_621843), Anti-rabbit IgG HRP-linked Antibody (1:200, Cell Signaling Technology, 7074P, AB_2099233).
7
Blue Native PAGE Analysis of Mitochondrial Proteins
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Blue Native gel electrophoresis (PAGE) was performed by isolating mitochondrial proteins from mutant and control cybrid cell lines as described elsewhere [21 (link)]. Samples containing 30 μg of proteins were separated on 3~12% Native PAGE gel. The primary antibodies applied for this experiment were NDUFA9 (Abcam, ab128744), SDHA (Abcam, ab14715), UQCRC2 (Abcam, ab203832), COX5A (Abcam, ab110262), and ATP5C (Abcam, ab119686). As a secondary detection antibody, an ECL system was used for band detection, after which densitometric calculations were performed as shown in Western blotting.
8
Western Blot Analysis of COX5a Protein
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Whole cell protein lysates were extracted using the Cellytic M buffer (Sigma-Aldrich) containing a Protease Inhibitor Cocktail (Sigma-Aldrich). Western blotting was performed according to previously described methods with minor modifications (Begum et al., 2013; Walker and Steinle, 2007) . 20µg of denatured protein was loaded onto a 4-20% Mini-Protean TGX Precast Protein gel (Bio-Rad, CA, USA) followed by semi-dry transfer to a nitrocellulose membrane. To measure the protein expression of COX5a in cells, a COX5a primary antibody (1:500-1:1000, #ab110262, Abcam) was used, as well as a secondary antibody-peroxidase conjugate for visualisation (Bio-Rad). The protein was visualised with chemiluminescence using a Clarity Western ECL kit (Bio-Rad) and images captured and analysed using a Chemidoc MP with Image Lab software (Bio-Rad). The expression of COX5a was normalized to GAPDH.
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