Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).
Anti opa1
Manufactured by BD
Sourced in United States
Anti-OPA1 is a laboratory reagent used to detect and quantify the presence of the OPA1 protein, which is involved in the regulation of mitochondrial morphology and function. It is a tool for researchers studying mitochondrial dynamics and cellular processes related to OPA1.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (7 mentions)
Anti mfn2, by Abcam (5 mentions)
Anti drp1, by BD (4 mentions)
Anti fis1, by Abcam (4 mentions)
Anti tom20, by Proteintech (4 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti phospho drp1 ser616, by Cell Signaling Technology (3 mentions)
Anti vdac, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti cytochrome c, by BD (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti mfn2, by Cell Signaling Technology (3 mentions)
Anti actin, by Merck Group (3 mentions)
Anti hsp90, by Cell Signaling Technology (2 mentions)
Anti dlp1, by BD (2 mentions)
Anti mfn2, by Merck Group (2 mentions)
Anti pgc 1α, by Abcam (2 mentions)
Anti yme1l, by Proteintech (2 mentions)
Anti p53, by Abcam (2 mentions)
29 protocols using anti opa1
1
Detailed Western Blot Methodology
2
Quantifying Mitochondrial Protein Expression
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15 to 70 μg of protein was electrophoresed on 10–15% vol/vol polyacrylamide SDS-PAGE gels. Proteins were electrophoretically transferred onto PVDF membranes. The membranes were subsequently blocked and, after blocking, were incubated at room temperature with the following antibodies: 1 : 500 anti-NOS1 (R-20) : sc-648, 1 : 500 anti-Mfn2 (H-68) : sc-50331, 1 : 2000 anti-actin (I-19) : sc-1616, and 1 : 2000 anti-VDAC1 (N-18) : sc-8828 were obtained from Santa Cruz, CA. 1 : 500 anti-OPA1 : 612607 and 1 : 1000 anti-DLP1 : 611113 were obtained from BD Biosciences. 1 : 1000 anti-phospho-DRP1 (Ser616) : #3455 was obtained from Cell Signaling. 1 : 1000 anti-nitrotyrosine, clone1A6 : #05-233 was obtained from EMD Millipore.
3
Western Blot Analysis of Mitochondrial Dynamics Proteins
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Primary hippocampal neurons were lysed in lysis buffer (10 mM Tris at pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton X-100, 1% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich) as previously described [40] (link). The extracted proteins were diluted into indicated amounts and separated on 8–12.5% SDS/PAGE and transferred onto polyvinylidene difluoride (PVDF) blotting membranes (BioRad). The membranes were blocked in 5% non-fat milk (Cell Signaling) dissolved in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 for 1 h at room temperature and probed with different antibodies: anti-DLP1 (1:3000, BD Biosciences), anti-phospho-Drp1 (Ser 616) (1:1000, Cell Signaling), anti-OPA1 (1:1000, BD Biosciences), anti-Mfn1 (1:1000, Novus Biologicals), anti-Mfn2 (1:1000, Sigma-Aldrich), anti-VDAC (1:1000, Cell Signaling), anti-cleaved-caspase-3 (Asp175) (1:1000, Cell Signaling), and anti-β-actin (1:10,000, Sigma-Aldrich). ECL (GE Healthcare) and Western Bright ECL (Advansta) were used to develop the film. Quantification analysis was performed on scanned films using Image J (NIH).
4
Mitochondrial Dynamics Protein Quantification
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The protein expression related to mitochondrial dynamics was measured by Western blot. The immunoblots were probed with anti-Opa1, anti-Drp1 (BD Biosciences), anti-Fis1 (Imgenex), anti-COX4, anti-MFN1 and Anti-MFN2 (Abcam) antibody overnight at 4°C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. Immunoblot results were visualized with ChemiDocXRS (Bio-Rad Laboratory) or LI-COR Odyssey® Infrared Imaging System. GAPDH was used as the internal loading control.
5
Kidney and Aorta Protein Analysis
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As previously described, proteins were extracted from the kidney22 and from the aorta.23 Electrophoresis was performed with 4% to 15% acrylamide Criterion gels (Bio‐Rad, Hercules, CA). Protein transfer onto nitrocellulose membranes (Bio‐Rad) was performed with the Trans‐Blot Turbo Transfer System (Bio‐Rad). Blots were blocked with Tris‐buffered saline–Tween‐BSA 5% and then incubated with the following primary antibodies: anti‐eNOS (endothelial nitric oxide synthase; BD Biosciences, Franklin Lakes, NJ; #610297), anti–phosphorylated eNOS (pS1177, BD Biosciences, #612393), anti‐CD45 (Abcam, Cambridge, UK; ab10558), anti‐NRF1 (Cell Signaling Technology, Danvers, MA; 46743s), anti‐UCP1 (Cell Signaling Technology; #14670), anti‐OPA1 (BD Biosciences; #612606), anti‐FIS‐1 (Santa Cruz Biotechnology, Dallas, TX; sc98900), anti‐p47 (BD Biosciences; #610355), anti‐gp91 (BD Biosciences; #611414) anti‐COX2 (BD Biosciences; #610204), and anti–β‐actin (Sigma, St. Louis, MO; A5316). Following incubation with a goat antirabbit IgG (H+L) or a goat antimouse IgG (H+L) secondary antibody, and the horseradish peroxidase conjugate (Thermo Fisher Scientific, Waltham, MA), the reaction was developed by enhanced chemiluminescence (Bio‐Rad) according to the manufacturer's instructions, and the signal was visualized by chemiluminescence.
6
Western Blotting of Oxidative Stress Markers
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Western blotting was performed using anti-4-hydroxynonenal (4-HNE; Abcam), anti-peroxiredoxin (Prx-SO3, Abcam), anti-MnSOD (Calbiochem, San Diego, CA), anti-copper-zinc superoxide dismutase (CuZnSOD; Chemicon, Temecula, CA), anti-histone H1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2)40 (link), anti-Opa1 (BD Bioscience, Franklin Lakes, NJ, USA), anti-Drp1 (Cell Signaling Technology, Danvers, MA, USA), anti-Bax (5B7; Santa Cruz), anti-Bcl-2 (Cell Signaling Technology), anti-cytochrome c (BD Bioscience), anti-cleaved caspase-3 (Merck Millipore, Darmstadt, Germany), anti-β-actin (Sigma), and anti-GAPDH (Novus, Littleton, CO, USA) antibodies.
7
Protein Immunoprecipitation and OPA1 Analysis
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Protein immunoprecipitation was performed using a Protein A/G immunoprecipitation kit (Biolinkedin, IK‐1004) in accordance with the instructions provided. Immune complexes were prepared from protein samples using anti‐acetylated lysine (CST, 9441S, 1:100), anti‐SIRT3 (CST, 5490S, 1:100), or control anti‐IgG (Proteintech, 30000‐0‐AP, 1:100). These complexes were bound to Protein A/G magnetic beads and subsequently collected. Western blotting was conducted using anti‐OPA1 (BD Biosciences, 612606, 1:1000), and band intensity was quantified.
8
Muscle Protein Quantification via Western Blot
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Total protein was extracted from TA muscle tissue, and western blotting was performed as described previously [23 (link)]. The following antibodies were used: anti-MSTN (ab203076, Abcam, MA, USA), anti-MuRF-1(ab172479, Abcam), anti-atrogin-1 (ab74023), anti-myoblast determination protein 1 (MyoD) (sc-377460, Santa Cruz Biotechnology, CA, USA), anti-myogenin (sc-52903, Santa Cruz Biotechnology), anti-TLR-9 (NBP2-24729; Novus Biologicals), anti-OPA-1(#612606, BD Biosciences), anti-PGC-1α (ab54481, Abcam), and anti-GAPDH (sc-365062, Santa Cruz Biotechnology). The band density was quantified using ImageJ software and normalized to GAPDH.
9
Phosphorylation Analysis of Parkin and PINK1
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Anti-GAPDH (1:1000 5% BSA/TBS-T, Cell Signaling),
anti-Parkin phospho-Ser65 (2 μg/mL, 5% milk/TBS-T, S210D, second
bleed, University of Dundee), anti-Parkin phospho-Ser65 (2 μg/mL,
5% milk/TBS-T, S210D, third bleed, University of Dundee), anti-pParkin
total (2 μg/mL, 5% milk/TBS-T, S966C, second bleed, University
of Dundee), anti-Parkin phospho-Ser65 (1:10,000 in 5% BSA/TBS-T, rabbit
monoclonal, MJF foundation), non-phosphopeptide Parkin Ser65 (2 mg/mL,
5% milk/TBS-T, University of Dundee), anti-PINK1 total (2 μg/mL,
5% milk/TBS-T, S085D, third bleed, University of Dundee), anti-Bcl-xL
total (1:1000, 5% BSA/TBS-T, Cell Signaling), anti-Bcl-xL phospho-Ser62
(1:1000, 5% BSA/TBS-T, Invitrogen), anti-PINK1 phospho-Thr257 (2 μg/mL,
5% milk/TBS-T, S114D, third bleed, University of Dundee), non-phosphopeptide
PINK1 Thr257 (2 mg/mL, 5% milk/TBS-T, University of Dundee), anti-OPA1
(1:1000, 5% BSA/TBS-T, BD Biosciences), anti-rabbit IgG HRP-linked
(1:1000 5% BSA/TBS-T, Cell Signaling), anti-sheep IgG HRP-linked (1:5000
5% milk/TBS-T, Abcam), and anti-mouse IgG HRP-linked (1:1000 5% BSA/TBS-T,
Cell Signaling) were used.
anti-Parkin phospho-Ser65 (2 μg/mL, 5% milk/TBS-T, S210D, second
bleed, University of Dundee), anti-Parkin phospho-Ser65 (2 μg/mL,
5% milk/TBS-T, S210D, third bleed, University of Dundee), anti-pParkin
total (2 μg/mL, 5% milk/TBS-T, S966C, second bleed, University
of Dundee), anti-Parkin phospho-Ser65 (1:10,000 in 5% BSA/TBS-T, rabbit
monoclonal, MJF foundation), non-phosphopeptide Parkin Ser65 (2 mg/mL,
5% milk/TBS-T, University of Dundee), anti-PINK1 total (2 μg/mL,
5% milk/TBS-T, S085D, third bleed, University of Dundee), anti-Bcl-xL
total (1:1000, 5% BSA/TBS-T, Cell Signaling), anti-Bcl-xL phospho-Ser62
(1:1000, 5% BSA/TBS-T, Invitrogen), anti-PINK1 phospho-Thr257 (2 μg/mL,
5% milk/TBS-T, S114D, third bleed, University of Dundee), non-phosphopeptide
PINK1 Thr257 (2 mg/mL, 5% milk/TBS-T, University of Dundee), anti-OPA1
(1:1000, 5% BSA/TBS-T, BD Biosciences), anti-rabbit IgG HRP-linked
(1:1000 5% BSA/TBS-T, Cell Signaling), anti-sheep IgG HRP-linked (1:5000
5% milk/TBS-T, Abcam), and anti-mouse IgG HRP-linked (1:1000 5% BSA/TBS-T,
Cell Signaling) were used.
10
Quantitative Analysis of OPA1 Expression
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Total RNA from fibroblasts was extracted with QIAmp RNA blood (Qiagen, Valencia, CA) and reverse transcribed, using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer's protocol. Expression levels of OPA1 were determined by quantitative real time PCR using SYBR Green I chemistry (Roche Diagnostics) and normalized on TUBB levels as the reference gene. Oligonucleotides sequences and PCR conditions are available upon request. cDNA was also sequenced as previously described.32 Thirty micrograms of cell lysates in radioimmunoprecipitation assay buffer (50mM Tris–Cl pH 7.6, 150mM NaCl, 1% NP‐40, 1% NaDOC, 0.1% sodium dodecyl sulfate [SDS], 5mM ethylenediaminetetraacetic acid [EDTA], 100µl/ml of protease inhibitor cocktail) were separated by 8% SDS–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio‐Rad Laboratories, Hercules, CA). The primary antibodies used were anti‐OPA1 (BD Biosciences, Franklin Lakes, NJ; O19820), anti–β‐tubulin (Sigma‐Aldrich, St Louis, MO; T6557), and anti‐VDAC (BioVision, Mountain View, CA; 3594). Detection and densitometry were performed with the Gel Logic 1500 imaging system (Eastman Kodak, Rochester, NY).
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