Cells were lysed (30 min at 4 °C), cleared by centrifugation, and protein concentrations were determined using BCA protein assay (Pierce). 15–30 μg protein lysate were separated on a 4–12% Bis–Tris gel (Invitrogen) and blotted onto a Nitrocellulose Membrane using iBlot (Invitrogen). Membrane was blocked for 1 h in Odyssey Blocking Buffer (LiCor, Lincoln, NE USA) followed by incubation with primary antibodies. IRDye® secondary antibodies (LiCor and Abcam, Cambridge, England) were used and signals were detected using the Odyssey Sa (LiCor). Following antibodies were used: MitoProfile® Total OXPHOS Human WB Antibody Cocktail (abcam #ab110411), human CD40 (abcam #13545), IL10 (#ab133575), IL6 (Cell Signaling, #12153), β-actin (abcam), α-Tubulin (LiCor) and β-tubulin (Santa Cruz, Dallas, TX, USA and abcam) were used as loading controls. Original Blot for Figure 2 and for the cut bands shown in Figure 3 B are presented in Appendix A (page 7).
Mitoprofile total oxphos human wb antibody cocktail
Manufactured by Abcam
Sourced in United States, United Kingdom
MitoProfile Total OXPHOS Human WB Antibody cocktail is a mixture of antibodies designed to detect the five subunits of the oxidative phosphorylation (OXPHOS) system in human samples. The cocktail includes antibodies specific to the NDUFB8, SDHB, UQCRC2, MT-CO2, and ATP5A proteins, which are part of the OXPHOS complexes I, II, III, IV, and V, respectively.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (6 mentions)
Chemidoc xrs imaging system, by Bio-Rad (5 mentions)
β actin, by Merck Group (5 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (3 mentions)
Mitotracker orange, by Thermo Fisher Scientific (3 mentions)
Mitotracker green, by Thermo Fisher Scientific (3 mentions)
Iblot, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
β tubulin, by Santa Cruz Biotechnology (2 mentions)
Irdye secondary antibody, by LI COR (2 mentions)
Imagequant, by GE Healthcare (2 mentions)
α tubulin, by LI COR (1 mentions)
Odyssey sa, by LI COR (1 mentions)
Il 10, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Acetylated lysine, by Cell Signaling Technology (1 mentions)
Sirt5, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
18 protocols using mitoprofile total oxphos human wb antibody cocktail
1
Western Blot Analysis for Protein Expression
2
Mitochondrial Protein Expression Analysis
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Aliquots containing 40 μg of proteins from each lysate cells were subjected to SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories; Hercules, CA, USA) using Trans Blot Turbo Transfer System. Membranes were probed with the following primary antibodies: MitoProfile Total OXPHOS Human WB Antibody cocktail, Pyruvate Dehydrogenase E1-alpha (PDH) and pPDHSer293 (1:500; Abcam Cambridge, UK), Acetylated Lysine (1:1000 Novus, UK), pAKTSer473, AKT, NAMPT, SIRT1, SIRT3, SIRT5 (1:1000; Cell Signaling Technology) and β-ACTIN (1:5000; SIGMA Aldrich, St. Louis, MO, USA). After incubation with corresponding suited horseradish peroxidase-conjugated secondary antibody (1:2500; Cell Signaling Technology) signals were developed using the enhanced chemiluminescence kit (ClarityTM Western ECL Substrate, Bio-Rad) and the ChemiDoc Imaging System XRS + (BioRad) and analysed with the Image Lab 4.1 software. The intensity of bands corresponding to Mitoprofile, Acetylated Lysine, NAMPT, SIRT1, SIRT3 and SIRT5 was normalized to the β-ACTIN signal while phosphorylated AKT and PDH was normalized to total protein.
3
Western Blot Analysis of OXPHOS Complexes
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HepG2 ρ0, HepG2 WT, or HepG2 cybrid cells were lysed using sonication and 10 µg of lysate protein was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–12% Bis-Tris gel (Invitrogen, UK) in MOPS buffer (MOPS; tris-base; 1.21% w/v, sodium dodecyl sulphate; 0.20% w/v, EDTA; 0.06% w/v in distilled water (dH20)).
This gel was then transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) in transfer buffer (tris-base; 0.30% w/v, glycine; 1.5% w/v, methanol; 20% v/v in dH20) and blocked using 10% non-fat dried milk in Tris Buffered Saline-Tween (TBS-T: TBS; 0.50% v/v, tween; 0.10% v/v in dH20).
Blocking solution was removed using TBS-T and the membrane was probed for CI-20, CII-30, CIII-core2, CIV-I, and CV-alpha subunits of complexes I-V of the electron transport chain using MitoProfile Total OXPHOS Human WB Antibody Cocktail (RRID:AB_2533836 , Cat No 45–8199, Abcam, Cambridgeshire, UK) (0.20% v/v in 10% non-fat dried milk in TBS-T). This was followed by anti-mouse secondary antibody (0.01% v/v in 10% non-fat dried milk in TBS-T) before visualization using an ECL system (GE Healthcare, Buckinghamshire, UK). For characterization data see Figure 9—figure supplement 2 .
This gel was then transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) in transfer buffer (tris-base; 0.30% w/v, glycine; 1.5% w/v, methanol; 20% v/v in dH20) and blocked using 10% non-fat dried milk in Tris Buffered Saline-Tween (TBS-T: TBS; 0.50% v/v, tween; 0.10% v/v in dH20).
Blocking solution was removed using TBS-T and the membrane was probed for CI-20, CII-30, CIII-core2, CIV-I, and CV-alpha subunits of complexes I-V of the electron transport chain using MitoProfile Total OXPHOS Human WB Antibody Cocktail (RRID:
4
Quantitative OXPHOS Protein Analysis
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Immunoblots were performed by standard protocol; PVDF membranes were probed with MitoProfile Total OXPHOS Human WB Antibody cocktail (1:500; Abcam Cambridge, UK) and β-ACTIN (1:5000; SIGMA Aldrich, St. Louis, MO, USA). The secondary antibody was horseradish peroxidase-conjugated (1:2500; Cell Signaling Technology). The signals were developed by an enhanced chemiluminescence kit (ClarityTM Western ECL Substrate, Bio-Rad), acquired by a ChemiDoc Imaging System XRS + (BioRad), and then analyzed for densitometry with the ImageJ Lab 4.1 software.
5
Western Blot Analysis of Adipocyte Proteins
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Preadipocytes and adipocytes were lysed (30 min at 4 °C), cleared by centrifugation and protein concentrations were determined using BCA protein assay (Pierce). 15 or 30 μg protein lysate were separated on a 4–12% BisTris gel (Invitrogen) and blotted onto a Nitrocellulose Membrane using iBlot (Invitrogen). Membrane was blocked for 1 h in Odyssey Blocking Buffer (LiCor, Lincoln, NE USA) followed by incubation with primary antibodies. Subsequently, IRDye® or AlexaFluor® secondary antibodies (LiCor or Abcam, Cambridge, England) were used and signals were detected using the Odyssey Sa or classic (LiCor). Following antibodies were use: MitoProfile® Total OXPHOS Human WB Antibody Cocktail (#ab110411, abcam), PFKP (D4B2, Cell Signaling), PKM2 (D78A4, Cell Signaling) and β-tubulin (Santa Cruz, Heidelberg, Germany or Abcam).
6
Whole-Cell Protein Analysis by Western Blot
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Cultured cells were lysed in 150 mM NaCl, 0,1% SDS, 5 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, 50 mM Tris, pH 7.4 and protease inhibitor cocktail and centrifuged at 12,000 g for 15 min at 4°C to remove cellular debris. The protein concentration was measured using the Bradford assay (Bio-Rad). The proteins were separated by 12% SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Bio-Rad). After blocking with 5% skim milk for 1 hour, the membranes were incubated with MitoProfile Total OXPHOS Human WB Antibody cocktail (1:500; Abcam Cambridge, UK), PDH-E1α (1:2,000; Abcam, Cambridge, UK), phospho-PDH-E1α (1:500; Abcam Cambridge, UK), Bcl-xl (1:1,000; Cell Signaling Technology), survivin (1:1,000; Cell Signaling Technology), GAPDH (1:2,500; Santa Cruz Biotech) and β-actin (1:5,000; SIGMA), overnight at 4°C. After incubation with corresponding suited 1:2.500 horseradish peroxidase-conjugated secondary antibody (1,2500; Cell Signaling Technology); the signals were developed using the enhanced chemiluminescence kit (ClarityTM Western ECL Substrate, Bio-Rad) and the ChemiDoc Imaging System XRS + (BioRad) and analysed with the Image Lab 4.1 software.
7
Immunoblotting Analysis of Cell Cycle Proteins
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Immunoblots were performed as previously described69 (link). Briefly, membranes were probed with the following primary antibodies: Cyclin D1 (1:1000; Cell Signaling Technology), Cyclin E (1:1000, EDM Millipore Temecula, CA, USA), Cdk1/2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (1:1000 Cell Signaling Technology, Danvers, MA, USA), p21/WAF1/Cip1 clone CP74 (1:1000, EDM Millipore Temecula, CA, USA), MitoProfile Total OXPHOS Human WB Antibody cocktail (1:500; Abcam Cambridge, UK) and β-Actin (1:5000; Sigma Aldrich, St. Louis, MO, USA). The secondary antibody was horseradish peroxidase-conjugated (1:2500; Cell Signaling Technology) and the signals developed by enhanced chemiluminescence kit (Clarity Western ECL Substrate, Bio-Rad), acquired by ChemiDoc Imaging System XRS + (BioRad) and analyzed for densitometry with the ImageJ Lab 4.1 software.
8
Protein Expression Analysis in CD4+ T Cells
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CD4+ T cell protein extracts (~ 30 μg) were boiled in Laemli sample buffer (Sigma-Aldrich) for 5 min, resolved on 10% SDS-polyacrylamide gels and transferred onto PVDF filters (Amersham). Membranes were blocked for 1 h in Tris-buffered saline (TBS), 0.05% Tween-20, 5% non-fat dry milk, followed by overnight incubation with specific primary antibodies diluted in the same buffer. The primary antibodies used are listed as follows: anti-GLUT1 (1:1000, Novus Biologicals), anti-MCT1 (1:1000, Novus Biologicals), anti-β-actin (1:8000, Sigma-Aldrich). Mitochondria-rich pellet (15 μg) was separated on a SDS-PAGE and, after blocking, membranes were incubated with MitoProfile total OXPHOS human WB antibody cocktail (1:1000, Abcam) and anti-Porin (1:1000, Santa Cruz). After washing with 0.1% Tween in TBS, membranes were incubated with a peroxidase-conjugated secondary antibody for 1 h, washed and developed using the ECL chemiluminescent detection system (Clarity™ Western ECL Substrate Biorad). The densitometric analyses of blots were performed by a computerized image processing system (Image J, 1.0 version).
9
Mitochondrial Protein Analysis by SDS-PAGE and Western Blot
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SDS-PAGE and Western blots were performed using 20 ug of total protein lysate as previously described [46 , 49 (link)]. Antibodies used were: rabbit anti-LC3B (Cell Signaling Technology, Danvers, MA), rabbit anti-phospho AMPK (Cell Signaling Technology, Danvers, MA), rabbit anti-actin (Sigma Aldrich, St. Louis, MI), Mitoprofile Total OXPHOS Human WB antibody cocktail (Abcam, Cambridge, MA), rabbit anti-pyruvate dehyrogenase (Cell Signaling Technology), rabbit anti phospho-pyruvate dehyrogenase E1 (S293) (Abcam) and anti-rabbit UCP2 (Abcam).
10
Western Blot Analysis of Mitochondrial Proteins
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Tissue samples were lysed as previously described [11] (link), and whole cell lysates from fibroblasts (15 µg of protein), and from muscle and liver (5 µg of protein), were separated by SDS-PAGE on a 4–12% polyacrylamide gel. A PVDF membrane was probed for 2 h at RT with 1∶1,000 MitoProfile Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam). Anti-mouse IgG HRP (GE Healthcare) at 1∶2,500 was used as the secondary antibody. Membranes were developed with ECL and exposed on Hyperfilm. Films were scanned on a Microtek ScanMaker 8700 and analysed using ImageQuant (GE Healthcare).
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