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.
Ab110242
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab110242 is a recombinant monoclonal antibody that recognizes PCNA (Proliferating Cell Nuclear Antigen). PCNA is a protein involved in DNA replication and repair processes.
Automatically generated - may contain errors
Lab products found in correlation
Ab14705, by Abcam (19 mentions)
Ab14745, by Abcam (16 mentions)
Ab14748, by Abcam (9 mentions)
Ab14714, by Abcam (9 mentions)
Ab14715, by Abcam (9 mentions)
Ab14734, by Abcam (8 mentions)
Ab110411, by Abcam (4 mentions)
Ab14730, by Abcam (4 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (3 mentions)
Ab14713, by Abcam (3 mentions)
Ab7291, by Abcam (2 mentions)
P0260, by Agilent Technologies (2 mentions)
P0399, by Agilent Technologies (2 mentions)
Ab14744, by Abcam (2 mentions)
Ab110258, by Abcam (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Ab32081, by Abcam (2 mentions)
Nitrocellulous membranes, by GE Healthcare (2 mentions)
32 protocols using ab110242
1
Western Blot Analysis of Skeletal Muscle
2
Western Blotting Analysis of Mitochondrial Complexes
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Total proteins were extracted using radioimmunoprecipitation assay buffer. Protein concentration was assessed with a Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The same amount of protein (μg) was loaded in each lane. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride or nitrocellulose membrane and subsequently subjected to immunoblotting analysis using appropriate antibodies. The amount of loading was further determined using a western blotting housekeeping protein (lamin A, 1:5000 dilution). Using a horseradish peroxidase-conjugated secondary antibody, protein bands on the blots were visualized with enhanced chemiluminescent western blot detection reagent. Antibodies including anti-tubulin (Abcam, ab7291), anti-LDHB (Abcam, ab75167), anti-complex I (Abcam, ab110242), anti-complex II (Abcam, ab14714), anti-complex III (Abcam, ab14745), anti-complex IV (Abcam, ab14705), anti-complex V (Abcam, ab14748) were purchased from commercial company (Abcam, Cambridge, UK).
3
Immunohistochemical Analysis of Mitochondrial Complexes
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Cryosections, 8‐μm thick, were cut and mounted on Superfrost Plus slides (J1800AMNZ, Thermo Scientific). Standard techniques were applied for enzyme histochemical analyses of COX, SDH and combined COX‐SDH [16 ]. For immunohistochemical studies of the mitochondrial respiratory chain complex, cryosections were fixed in 4% formaldehyde at 4°C for 10 min, washed in Tris‐buffered saline‐Tween 20 (TBS‐T) for 10 min, permeabilised in a graded methanol series (70% 10 min, 95% 10 min, 100% 20 min, 95% 10 min and 70% 10 min) and washed in TBS‐T for 5 min. For the study of MHC‐class I (HLA‐ABC), the cryosections were fixed with acetone. The tissue sections were processed in a Dako Autostainer using the EnVision FLEX DAB+ Substrate Chromogen System kit and incubated with the following primary antibodies for 1 h: anti‐NDUFB8 (complex I, ab110242, Abcam, UK, 1:100), anti‐SDHB (complex II, ab14714, Abcam, 1:500), anti‐MTCO1 (complex IV, ab14705, Abcam, 1:2000), anti‐VDAC1 (porin, a mitochondrial marker, ab14734, Abcam, 1:2000) and anti‐human HLA‐ABC (M0736, clone W6/32, Dako, 1:1000). These antibodies to subunits of complex I, II and IV of the respiratory chain have been well characterised and demonstrated to be reliable tools in studies on mitochondrial myopathies [17 (link), 18 (link), 19 (link)].
4
Profiling Mitochondrial Protein Levels
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The following primary antibodies were used in this study: MS601 (ab110411, Abcam, 1:1000 dilution), NDUFB8 (ab110242, Abcam, 1:1000 dilution), SDHA (ab14715, Abcam, 1:2000 dilution), UQCRC2 (ab14745, Abcam, 1:1000 dilution), COXI (ab14705, Abcam, 1:1000 dilution), ATP5A (ab14748, Abcam, 1:1000 dilution), and β-actin (Cloud Clone Corp. CAB340Hu22, 1:10 000 dilution). HRP-conjugated mouse secondary antibody was used (DAKO, P0260, 1:2000 dilution), followed by detection using the ECL (GE Healthcare) and BioRad imaging system (Image Lab).
5
Protein Expression Analysis via Western Blot
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Cells were scraped on ice in RIPA buffer (Millipore), containing protease and phosphatase inhibitors (Roche). Proteins were separated in 4%–20% SDS-PAGE gels and transferred onto nitrocellulous membranes (GE Healthcare). After blocking in 5% dry milk in TBST, membranes were incubation with the following primary antibodies and dilutions overnight at 4°C: anti-COX7A2L (Protein-tech, 11416, 1:500), anti-HIF1α (BD Biosciences, 610959, 1:500), anti-GOT1 (Novus Biologics, NBPI-54778, 1:500), anti-GOT2 (Novus Biologics, NBP1–47469, 1:500), anti-ERK2 (Abcam, ab32081, 1:1,000), anti-NDUFB8 (Abcam, 1:1,000, ab110242), anti-UQCRC2 (1:1,000, ab14745, Abcam), anti-MTCO1 (1:1,000, ab14705, Abcam) and anti-Actin (Sigma, A5441, 1:5,000). Membranes were washed with TBST, incubated with the appropriate peroxidase-conjugated secondary antibody (Cell Signaling Technologies, 1:5,000, 7045S or 7076S) for 1 h and developed using the enhanced chemiluminescence (ECL) detection system (Bio-Rad, 1705061) using a ChemiDoc (Bio-Rad).
6
Isolation and Analysis of Mitochondrial Complexes
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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.
7
Mitochondrial Protein Expression Analysis
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Lipoic acid (437695, Calbiochem/Merck, Darmstadt, Germany); Hsp60 (AB1-SPA-807-E, Enzo Lifesciences Farmingdale, US); Human OXPHOS cocktail (ab110411, Abcam, Cambridge, UK; 1:1000); VDAC1 (ab14734, Abcam; 1:5000); SLC25A26 (ab175209, Abcam; 1:1000); GAPDH (600004, Proteintech; 1:5000) SDHA (ab14715, Abcam; 1:1000), COXI (ab14705, Abcam; 1:1000); NDUFB8 (ab110242, Abcam; 1:1000); UQCRC2 (ab14745, Abcam; 1:1000); ATP5A (ab14748, Abcam; 1:1000). Secondary antibodies used include Polyclonal Donkey Anti-Rabbit Ig/HRP (NA9340V, SigmaAldrich, St. Louis, US); Polyclonal Sheep Anti-Mouse Ig/HRP (NA9310V, SigmaAldrich); Polyclonal Rabbit Anti-Mouse Ig/HRP (P0161, Dako/Agilent; 1:2000) and Polyclonal Swine Anti-Rabbit Ig/HRP (P0399, Dako; 1:3000).
8
Western Blot Analysis of Liver Proteins
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Liver samples were homogenised in buffer consisting of 10 mM Tris–HCl pH 7.5, 1 mM EGTA, 75 mM sucrose, 225 mM sorbitol and 0.1% BSA, before lysis at 4°C in 50 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS and 1 × protease inhibitor cocktail (Roche). Protein concentration was determined by Lowry assay (DC Reagent, Bio-Rad), and 5 μg of protein resolved on PAGE gels (Novex) before transfer to Imobilon-P membrane (Millipore). Following blocking with 5% non-fat dry milk in PBS with 0.1% (v/v) Tween-20, membranes were incubated overnight with the following primary antibodies: mouse anti-NDUFB8 (1:10 000, Abcam AB110242), mouse anti-COX IV (1:10 000, Abcam AB14744), rabbit anti-MPV17 (1:1000, Proteintech 10310-1-AP) or rabbit anti-TOM20 (1:20 000, Santa Cruz Biotechnology SC-11415). Membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:4000, Promega) in 5% non-fat dry milk in PBS with 0.1% (v/v) Tween-20, and visualised using enhanced chemiluminescence (ECL, G.E. Healthcare).
9
Protein Expression Analysis in Mitochondria
10
Multimodal Analysis of Metabolic and Proliferative Changes in Liver and Kidney
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The histological changes were analyzed in liver and kidney tissues. Ki67 staining was performed in liver tissue. For western blot, the tissues were homogenized in radioimmunoprecipitation assay buffer (RIPA) with a tissue disruptor. The RIPA buffer contained 150 mM sodium chloride; 1.0% NP–40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulphate; 50 mM Tris, pH 8.0. An amount of 20 μg total protein was loaded for western blot analysis. The primary antibodies used were anti-MTCOI antibody (Abcam: ab14705) for mtDNA encoded peptide in ETC complex IV; anti-SDHA antibody (Abcam; ab14715) and anti-NDUF8 antibody (Abcam: ab110242) were used to nuclear encoded proteins in ETC. Anti-Ki-67 (Abcam: ab92742) was used to detected cell proliferation in liver. Anti-Phosphoserine Aminotransferase (Abcam: ab96136), anti-PHGDH/Malate dehydrogenase (Abcam: ab240744), anti-SHMT1(Abcam: ab186130) and anti-MTHFD1(Abcam: ab226341) were used to investigate serine-folate 1C metabolic pathway. Mouse anti-beta actin (Sigma: A5441) or anti-VDAC1/Porin (Abcam: ab14734) was used as loading control. The secondary antibodies are polyclonal rabbit anti-mouse IgG (Dako: P0260) and donkey anti-rabbit (Santa Cruz: SC2313).
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