Antibodies used for western blot and immunostaining were mouse anti-Flag (1:1,000, Sigma-Aldrich, F1804), rabbit anti-Flag (1:1,000, Sigma-Aldrich, F7425), mouse anti-TOM20 (1:1,000, Santa Cruz, sc17764), mouse anti-Tom40 (1:1000, Santa Cruz, sc365467), mouse anti-Tom70 (1:1000, Santa Cruz, sc390545), mouse anti-VDAC1 (1:1000, Santa Cruz, sc390996), rabbit anti-cytochrome C (1:1000, Abcam, ab90529), rabbit anti-C-I30 (1:1000, Abcam, ab14711), mouse anti-Core2 (1:1000, Santa Cruz, sc390378), mouse anti-OPA1 (1:1000, BD Biosciences 612806), rat anti-HA (1:1000, Roche, 3F10), mouse anti-actin (1:5000, Sigma, A2228), rabbit anti-mitofilin (1:1000, Abcam, ab48139), rabbit anti-CHCHD3 (1:1000, Abcam, ab98975), mouse anti-ApooL (1:1000, Santa Cruz, sc-390958), mouse anti-LETM1 (1:1000, Abcam, ab55434), mouse anti-Myc (1:1000, Santa Cruz, 9E10), rabbit anti-Minos1 (1:1000, Abcam, ab84969), rabbit anti-fly-Opa1(1:1000, Sigma, M6319). Antibodies for dot blot: rat anti-poly (GR) (1:500, Millipore, MABN778), rabbit anti-poly(GA) (1:500, Proteintech, 24492–1-AP), rabbit anti-ATP6 (1:1000, abcam, AB102573), mouse anti-Flag (1:1000, Sigma, F1804), mouse anti-actin (1:3000, Sigma, A2228).
Mouse anti opa1
Manufactured by BD
Sourced in United Kingdom, United States
Mouse anti-OPA1 is a primary antibody that specifically binds to the OPA1 protein. OPA1 is a protein involved in the regulation of mitochondrial fusion and cristae organization. This antibody can be used for the detection and analysis of OPA1 in various biological samples and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti myc, by Santa Cruz Biotechnology (2 mentions)
Mouse anti tom70, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti flag, by Merck Group (2 mentions)
Mouse anti vdac1, by Santa Cruz Biotechnology (2 mentions)
Mouse anti tom20, by Santa Cruz Biotechnology (2 mentions)
Mouse anti tom40, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti c i30, by Abcam (2 mentions)
Mouse anti core2, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti mitofilin, by Abcam (2 mentions)
Rabbit anti chchd3, by Abcam (2 mentions)
Mouse anti apool, by Santa Cruz Biotechnology (2 mentions)
Mouse anti letm1, by Abcam (2 mentions)
Rabbit anti minos1, by Abcam (2 mentions)
Rabbit anti fly opa1, by Merck Group (2 mentions)
Rat anti poly gr, by Merck Group (2 mentions)
Rabbit anti atp6, by Abcam (2 mentions)
Rabbit anti cytochrome c, by Abcam (2 mentions)
Mouse anti actin, by Merck Group (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Rat anti ha, by Roche (2 mentions)
13 protocols using mouse anti opa1
1
Mitochondrial Protein Immunodetection Protocol
2
Mitochondrial Protein Immunodetection Protocol
3
Western Blot Analysis of Mitochondrial Proteins
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Protein concentration was determined using the bicinchoninic acid method. A total of 65 µl of each sample was mixed with 25 µl of NuPAGE LDS 4 × sample buffer (ThermoFisher Scientific, NP0007) and 10 µl of NuPAGE Sample Reducing Agent (ThermoFisher Scientific, NP0004) and heated at 70 °C for 10 min. Samples were run on 4–12% NuPAGE Bis–Tris gels (Invitrogen) and transferred to reinforced nitrocellulose membranes (Bio-Rad). After blocking with 5% fat-free milk in TBST buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.6) for 60 min at room temperature, the membranes were incubated overnight with the following primary antibodies: rabbit anti-phospho-DRP1 (1:1000 dilution, Ser637, Cell Signaling, 4867), mouse anti-OPA1 (1:1000 dilution, BD Bioscience, 612,606), rabbit anti-FIS1 (1:500 dilution, FL-152, Santa Cruz, sc-98900), and mouse anti-VDAC1 (1:500 dilution, B-6, Santa Cruz, sc-390996). After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG antibody (1:2000 dilution, Vector, PI-1000) or peroxidase-labeled horse anti-mouse IgG antibody (1:4000 dilution, Vector, PI-2000). Immunoreactive species were visualized using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, 34,580) and an LAS 3000 cooled CCD camera (Fujifilm, Japan).
4
Mitochondrial Dynamics Protein Detection
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Cell lysate was obtained by incubating cells with ice-cold RIPA buffer containing protease inhibitors, and mitochondria fractionation was performed using the Thermo Scientific Mitochondria Isolation Kit for Cultured Cells per manufacturer's instructions. Cell lysate or mitochondrial protein (∼10 μg) was loaded into polyacrylamide gels (Bio-Rad Laboratories) and separated by SDS-PAGE.
The following primary antibodies were used: mouse anti-Drp1 (Catalog #611112, BD Biosciences), mouse anti-OPA1 (#612 606, BD Biosciences), rabbit anti-MFN1 (#14 739) and anti-MFN2 (#9482, Cell Signaling Technology), rabbit antiphospho-DRP1-Ser616 (#4494) and anti-phospho-DRP1-Ser637 (#6319, Cell Signaling Technology), rabbit anti-VDAC (#4661, Cell Signaling Technology) and rabbit anti-GAPDH (#ABS16, Sigma-Aldrich). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were used as secondary antibodies. Primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:5000. SNO-Drp1 was detected using a Pierce S-Nitrosylation Western Blot Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Protein expression was visualised with Western ECL Blotting Substrates (Bio-Rad), and image acquisition was performed using a Bio-Rad ChemiDoc MP Imaging System. Densitometry was performed using ImageJ software (NIH).
The following primary antibodies were used: mouse anti-Drp1 (Catalog #611112, BD Biosciences), mouse anti-OPA1 (#612 606, BD Biosciences), rabbit anti-MFN1 (#14 739) and anti-MFN2 (#9482, Cell Signaling Technology), rabbit antiphospho-DRP1-Ser616 (#4494) and anti-phospho-DRP1-Ser637 (#6319, Cell Signaling Technology), rabbit anti-VDAC (#4661, Cell Signaling Technology) and rabbit anti-GAPDH (#ABS16, Sigma-Aldrich). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were used as secondary antibodies. Primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:5000. SNO-Drp1 was detected using a Pierce S-Nitrosylation Western Blot Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Protein expression was visualised with Western ECL Blotting Substrates (Bio-Rad), and image acquisition was performed using a Bio-Rad ChemiDoc MP Imaging System. Densitometry was performed using ImageJ software (NIH).
5
Immunoblotting of Cellular Proteins
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Cells were collected, washed with PBS, and then lysed in cell disruption buffer (Ambion) containing inhibitors of phosphatases and proteases (PhosphoSTOP and Complete, Roche). Proteins were separated by SDS/PAGE in 4–20% Tris-HEPES gradient gels (BioRad), and transferred to nitrocellulose membranes (GE Healthcare). The membranes were saturated with 3% bovine serum albumin prepared in TBS (50 mM TRIS-Cl, pH 7.5, 150 mM NaCl)-0.01% Tween-20 and then incubated overnight with the following antibodies: mouse anti-OPA1 (BD Biosciences, 1:1000), mouse anti-G6PD (Santa Cruz Biotechnology, 1:1000), rabbit anti-phospho-4EBP (Cell Signaling Technology, 1:1000), rabbit anti-non phospho-4EBP (Cell Signaling Technology, 1:1000), rabbit anti-PARP (Cell Signaling Technology, 1:1000) and rabbit anti-GAPDH (GeneTex International Corp., 1:10000). Membranes were then washed twice and incubated for 1 h with an HRP-conjugated anti-mouse or anti-rabbit antibody (Thermo Fisher Scientific, 1:5000). Chemiluminescent signals were detected using Lite Ablot Turbo (EuroClone) and a Cambridge UVITEC imaging system.
6
Protein extraction and Western blot
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Total cell lysates were generated in RIPA buffer (1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS in PBS). Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to manufacturer’s instructions. Westerns were performed as described [30] (link). PVDF was immunoblotted with antibodies against HTRA2 (R&D Systems), HSP90 or β-actin (Santa Cruz), OPA1 (mouse anti-OPA1 from BD Pharminogen, rabbit anti-OPA1 from Abcam), VDAC, MFN2, LC3β, P62 or PHB2 (Cell Signaling). ImageJ was used to quantify band intensity to calculate OPA1 processed form ratios from at least three animals per genotype.
7
Quantifying Mitochondrial Dynamics in Neural Stem Cells
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When animal drug administration was over, half of the mice in each group were xed with paraformaldehyde while the rest of their SVZ was dissected under a microscope, and then total protein was extracted. Brain tissues were dissolved in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, with 150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA sterile solution; Lugen Sci, Korea) plus protease inhibitor cocktail (Sigma). The lysates were centrifuged at 4℃ for 20 min (14,000 g), and supernatants were transferred to fresh tubes. Brie y, 30ug of protein was separated by SDS-gel electrophoresis and transferred to hydrophobic polyvinylidene di uoride (PVDF) membranes (GE Healthcare, Little Chalfont, UK). The membranes were blocked in 5% skim milk in PBST. Membranes were probed with the following primary antibodies: mouse anti-Mfn1 (Abcam), rabbit anti-Mfn2 (Epitomics), mouse anti-OPA1 (BD biosciences), rabbit anti-Fis1 (Abnova), mouse anti-Dlp1 (BD biosciences), mouse anti-nestin (Millipore), and mouse anti-Actin (santacruz). As secondary antibodies, 1:5000 dilutions of horeseradish peroxidase-conjugated goat anti-rabbit antibody (Solarbio) and anti-mouse antibody (Solarbio) were used. Antigen-antibody complexes were visualized with ECL solution (GenDEPOT). For quantitative analysis, immunoblotting band densities were measured by image J 50 .
8
Quantitative Analysis of Mitochondrial Dynamics
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C17.2 and HeLa cells were harvested in Hepes Buffer A (50 mM HEPES (pH 7.5)), 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM EDTA, and protease inhibitors (Merck) containing 1% Triton X-100, incubated on ice for 20 min and sonicated. The lysate was centrifuged to remove insoluble material, and the protein concentration was determined using Bradford assay according to the manufacturer’s instructions (Biorad, Watford, UK). Absorbance was measured at 590 nm, and protein concentration was interpolated from a bovine serum albumin standard curve.
Protein lysates (50 μg) were resolved on 4–12% Bis-Tris SDS-page gels in NuPage MOPS buffer (Thermo Fisher Scientific, Loughborough, UK) and transferred to polyvinylidene fluoride membrane PVDF membrane (Immobilon FL, Merck, Gillingham, UK) specialized for low fluorescent background. Western blot and LI-COR fluorescence imaging were carried out at 680/800 nm using an Odyssey DLx imager (LI-COR Biosciences, Cambridge, UK) as described previously [14 (link)]. The following antibodies were used in this study: mouse anti-OPA1 (Clone 18/BD Bioscience 612607), mouse anti-MFN2 ([6A8], Abcam, Cambridge, UK), anti-mouse GAPDH (Merck G8795), IRDye 680RD and 800CW goat anti-mouse secondary antibodies (LI-COR Biosciences). Densitometric quantification of OPA1, MFN2, and GAPDH was performed using Image Studio software v5.2 (LI-COR Biosciences).
Protein lysates (50 μg) were resolved on 4–12% Bis-Tris SDS-page gels in NuPage MOPS buffer (Thermo Fisher Scientific, Loughborough, UK) and transferred to polyvinylidene fluoride membrane PVDF membrane (Immobilon FL, Merck, Gillingham, UK) specialized for low fluorescent background. Western blot and LI-COR fluorescence imaging were carried out at 680/800 nm using an Odyssey DLx imager (LI-COR Biosciences, Cambridge, UK) as described previously [14 (link)]. The following antibodies were used in this study: mouse anti-OPA1 (Clone 18/BD Bioscience 612607), mouse anti-MFN2 ([6A8], Abcam, Cambridge, UK), anti-mouse GAPDH (Merck G8795), IRDye 680RD and 800CW goat anti-mouse secondary antibodies (LI-COR Biosciences). Densitometric quantification of OPA1, MFN2, and GAPDH was performed using Image Studio software v5.2 (LI-COR Biosciences).
9
Immunoblotting of Mitochondrial Proteins
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Collected cells were lysed with RIPA buffer (Thermo Fisher Scientific, 89900) containing a complete protease inhibitor cocktail (Roche) at 4°C for 20 min with shaking, and then centrifuged at 4°C for 20 min at 13,000 rpm. The supernatant was collected, the total proteins were quantified, and 10 μg of total protein from each sample was boiled for 10 min in sample buffer (BioRad, #161-0737), resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with TBST containing 5% skim milk, incubated with primary antibodies in TBST containing 5% skim milk, and incubated with HRP-conjugated secondary antibodies. The results were visualized with ECL reagents. The following primary antibodies were used: mouse anti-Opa1 (BD, 612606), rabbit anti-Hsp60 (CST, 12165), rabbit anti-Bcl2 (CST,3498), and rabbit anti-β-actin (CST, 4967S).
10
Protein Expression Analysis by Western Blot
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Equal amount of total protein samples were separated by SDS/PAGE (12% Bis-Tris gel; Life Technologies), and then transferred to PVDF membrane (BioRad Laboratories). After blocking in TBST buffer (20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween-20) containing 5% (wt/vol) nonfat dry milk (Santa Cruz) for 1 h at room temperature, the membrane was then incubated with primary antibodies overnight at 4°C. This was followed by incubation with the corresponding secondary antibody for 1h at room temperature. The following antibodies were used: mouse anti-Parkin antibody (1:500; Santa Cruz), rabbit anti-LC3B (1:1000; Cell Signaling Technology), rabbit anti- β-amyloid (1:6000; Cell signaling Technology), rabbit anti-TOM40 (1:1000; Santa Cruz), rabbit anti- MFN2 (1:6000; Cell signaling Technology), mouse anti-OPA1 (1:5000; BD Transduction Lab), mouse anti- DLP1 (1: 2000; BD Transduction Lab), and goat anti-mouse IgG HRP conjugated and goat anti-rabbit IgG HRP conjugated (1:6000; Life Technologies). Image J software (National Institutes of Health) was used to analyze the scanned blots and to quantify protein signal intensity.
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