Immunoblotting was performed to quantify the expression levels of various proteins in brain mitochondrial lysates [20 (link)]. Samples were prepared by lysing mitochondrial fractions with RIPA buffer as previously described [19 (link)]. Proteins were separated in 12% Bis-Tris Gel (NuPAGE, Life Technologies) and then transferred to PVDF membrane (ImmunBlot Membrane, BioRad). Membranes were blocked with 5% non-fat dry milk (Labscientific Inc.) for 1 h at room temperature and probed with appropriate primary antibodies overnight at 4°C; followed by the incubation with the appropriate secondary antibody for 1 h at room temperature. Proteins were detected using ECL (Clarity Substrate, BioRAD) and imaged using a Chemidoc system (BioRAD). The following antibodies were used in the experiments: anti-Cyclophilin D (Calbiochem, 1:5,000), anti-Tom 40 (Santa Cruz, 1:500), and for all the major F1FO ATP synthase subunits: anti-OSCP (Santa Cruz Biotechnologies, 1:5,000), anti-ATP5A (Santa Cruz Biotechnologies, 1:5,000), anti-ATP5B (Santa Cruz Biotechnologies, 1:5,000), anti-ATP6 (Protein-Tech, 1:5000), anti-ATP5F1 (Santa Cruz Biotechnologies, 1:500), anti-ATP5C1 (Santa Cruz Biotechnologies, 1:500), and anti-ATP synthase C (Abcam, 1:5000). The intensities of the immunoreactive bands were analyzed using NIH ImageJ software.
Anti atp5a
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-ATP5A is a laboratory reagent used for the detection and quantification of the ATP synthase subunit A (ATP5A) in biological samples. It functions as a specific antibody that binds to the ATP5A protein, allowing researchers to study its expression and localization within cells.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc system, by Bio-Rad (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Anti oscp, by Santa Cruz Biotechnology (1 mentions)
Anti tom40, by Santa Cruz Biotechnology (1 mentions)
Anti atp5b, by Santa Cruz Biotechnology (1 mentions)
Immunblot membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti hur, by Santa Cruz Biotechnology (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Anti β actin, by Abcam (1 mentions)
3 protocols using anti atp5a
1
Quantifying Mitochondrial Protein Levels
2
Western Blot Analysis of Protein Markers
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Western blot analysis was performed as reported previously [30 (link)]. Briefly, total cell lysates were prepared in RIPA buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Nonidet P-40, 1 mM EDTA, and 0.1% (w/v) SDS). Protein samples were separated on 10% SDS-polyacrylamide (SDS-PAGE) gels, and transferred onto Nitrocellulose membranes (Invitrogen iBlot Stack, Carlsbad, CA, USA). The membranes were incubated with a specific primary antibody in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) and 5% nonfat dry milk at 4 °C for overnight. After three washes with TBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA) for 1 h at room temperature, immunoblots were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). The following antibodies were purchased from Millipore (Burlington, MA, USA): anti-AUF1, Anti-HuR, anti-beta-Actin, anti-GAPDH, and anti-ATP5A were purchased from Santa Cruz Biotechnology (Dallas, TA, USA).
3
Antibodies for Mitochondrial Protein Analysis
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The antibodies that were used in the present study included: anti-adenylate kinase 2 (Ak2; catalog number: PA5-49482; Invitrogen, Waltham, MA, USA), anti-porin (catalog number: 185-197; Sigma-Aldrich, St. Louis, MO, USA), anti-cytochrome c oxidase subunit IV (COX4; catalog number: ab16056; Abcam, Cambridge, UK), anti-apoptosis-inducing factor (AIF; catalog number: MA5-15880; Invitrogen, Carlsbad, CA, USA), anti-ATP5A (catalog number: sc-136178; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (catalog number: ab6276; Abcam) antibodies. The anti-ES1 antibody was generated in our laboratory, as previously reported [29] (link). Keyhole limpet hemocyanin conjugated to the C-terminal 17 amino acid peptide of mouse ES1 (Cys-HDGIGAMVKNVLELTGK) was used as the antigen for antibody production.
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