Supernatant samples were separated under denaturing conditions in 12.5% SDS-PAGE gels and transferred to a nitrocellulose membrane. Precision Plus Pre-stained Standards from Bio-Rad Laboratories (Hercules, CA, USA) were used as markers. After overnight blocking, the membrane was incubated with the appropriate primary antibody. Anti-melatonin related receptor antibody (rabbit polyclonal) was used at the 1:1000 dilution (abcam, USA). The monoclonal anti-CNP antibody (anti-CNP Ab) was obtained as described [44 (link)] and used at the 1:10,000 dilution, the monoclonal anti-cytochrome c antibody (# DLN 06724 from Dianova; Hamburg, Germany) was used at the 1:2000 dilution, and the Tom20 antibody (Cell Signaling, USA) was used to rule out the contamination of the supernatant with mitochondria and as a loading control (1:1000 dilution). The immunoreactivity was detected using the appropriate secondary antibody conjugated to horseradish peroxidase (Jackson Immuno Research, West Grove, PA, USA). The peroxidase activity was detected using ECL chemiluminescence reagents (Pierce, Rockford, IL, USA).
Tom20 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Tom20 antibody is a laboratory research tool used to detect the Tom20 protein, which is a component of the translocase of the outer membrane (TOM) complex in mitochondria. The Tom20 protein functions as a receptor that recognizes and binds to mitochondrial preproteins, facilitating their import into the mitochondrial matrix. The Tom20 antibody can be used in various techniques, such as Western blotting and immunofluorescence, to study the localization and expression of the Tom20 protein in biological samples.
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Lab products found in correlation
Secondary antibody conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Precision plus pre stained standards, by Bio-Rad (1 mentions)
Hrp linked anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Hrp linked antimouse secondary antibody, by Cell Signaling Technology (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Anti tfam antibody, by Santa Cruz Biotechnology (1 mentions)
Total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Accuri cflow software, by BD (1 mentions)
Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Bx51 dp71, by Olympus (1 mentions)
Mito tracker green, by Beyotime (1 mentions)
6 protocols using tom20 antibody
1
Western Blot Analysis of Protein Targets
2
Mitochondrial Protein Expression Analysis
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MCU antibody; Sigma-Aldrich, Cat. no. HPA016480-100UL (1:2,000)
DYKDDDDK Tag Rabbit antibody; Cell Signaling Technology, Cat. no. 14793S (1:3,000)
EMRE antibody; Bethyl Laboratories, Cat. no. A300-BL19208 (1:1,000)
ATP5A antibody; Abcam Biochemicals, Cat. no. ab14748 (1:5,000)
TOM20 antibody; Cell Signaling Technology, Cat. no. 42406S (1:5,000)
HRP-linked antirabbit secondary antibody; Cell Signaling Technology Cat. no. 7074S (1:10,000)
HRP-linked antimouse secondary antibody; Cell Signaling Technology Cat. no. 7076S (1:10,000)
3
Mitochondrial Distribution in Primary Astrocytes
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Primary cortical astrocytes grown on poly-d -lysine-coated coverslips were fixed with 20% methanol for 7 min and permeabilized with 0.1% Triton X‐100/PBS for 15 min. The cells were then incubated in 5% bovine serum albumin (BSA)/PBS blocking solution for 30 min and subsequently incubated with a Tom20 antibody (Cell Signaling, 42406S) diluted in blocking solution overnight at +4°C. After washing, the fluorescent Cy3-conjugated secondary antibody (Jackson ImmunoResearch) were diluted in 1% BSA/PBS and applied for 1 h at room temperature. Immunofluorescence data were obtained using Zeiss Axio Scope.A1 fluorescence microscope with a Zeiss AxioCam MRm Camera (Carl Zeiss) equipped with Zen 2011 Blue software. To estimate mitochondria distribution in the cells the number of cells with clear mitochondrial staining in distal processes (phenotype 1) and the number of cells with distal processes virtually devoid of any staining (phenotype 2) were quantified as percent to the total number of cells.
4
Immunoblotting Analysis of Mitochondrial Proteins
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The left ventricle specimens for immunoblotting were lysed in STE buffer (320 mM sucrose, 10 mM Tris–HCl, 5 mM EDTA, 50 mM NaF, 2 mM Na3VO4, and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)), and the lysates were subjected to SDS–polyacrylamide gel electrophoresis. Immunoblotting was then performed with voltage-dependent anion channel 2 (VDAC2) antibody (#9412, Cell Signaling Technology, Beverly, MA, USA), the translocase of the outer mitochondrial membrane 20 (TOM20) antibody, (#42406, Cell Signaling Technology), total OXPHOS Rodent WB antibody cocktail (ab110413, Abcam), anti-PGC-1 polyclonal antibody (#AB3242, EMD Millipore Corporation, CA, USA), anti-NRF1 antibody (sc-33771, Santa Cruz Biotechnology, Inc.), anti- TFAM antibody (sc-23588, Santa Cruz Biotechnology, Inc.), anti-PPARα monoclonal antibody (Santa Cruz Biotechnology, Inc.), anti-ATF5 rabbit monoclonal antibody (Abcam), anti-CHOP mouse monoclonal antibody (#2895, Cell Signaling Technology), and anti-actin antibody (A2066, Sigma-Aldrich, St. Louis, MO, USA). Antigens were visualized by peroxidase-bonded anti-Mouse or rabbit IgG secondary antibodies (Promega Corporation, USA) and enhanced chemiluminescence reagents (Thermo Fisher Scientific, USA). The band densities were quantified by CS analyzer 4 image analyzing software (Atto, Tokyo, Japan).
5
Mitochondrial Mass Cytofluorometric Evaluation
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Mitochondrial mass was evaluated by cytofluorometry using Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific, Inc, # 88-8824-00.) according to a two-step protocol for intracellular proteins provided by the manufacturer. Forty-eight hours after transient transfection, K562 cells were harvested and washed twice with cold PBS by centrifugation at 3000 rpm for 10 min at room temperature. Cells were incubated for 30 min with Tom 20 antibody (1:200 dilution Cell Signaling, #42406) at room temperature and protected from light. Cells were then washed and incubated in PBS with a goat anti-rabbit IgG-FITC antibody (1:400 dilution, Alexa Fluor 488 #A11034) for 30 min at room temperature and protected from light. Stained cells were resuspended in an appropriate volume of Flow Cytometry Staining Buffer and the mean fluorescence intensity (MFI) was determined by flow cytometry using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) and BD Accuri C-flow software. Net fluorescence signals were evaluated after IgG (Sigma-Aldrich) background subtraction.
6
Ginsenoside CK Induces Mitophagy
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In order to research mitophagy activated by ginsenoside CK, we transfected mCherry LC3 plasmid into C2C12 cells using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scienti c, L3000015). Then the cells were incubated with 0.25 mM FA or FA plus 7.5 µM ginsenoside CK for 24 h. Subsequently, Mito-Tracker Green (Beyotime, C1048) was used to stain intracellular mitochondria, and nally, uorescence microscope (Olympus, BX51/DP71) was used to observe.
To observe the co-localization of Parkin and Tom20, GFP-Parkin plasmids were transfected into C2C12 cells by Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scienti c, L3000015). Then the cells were treated with 20 µM carbonyl cyanide-interchlorophenhydrazone (CCCP), CCCP plus 7.5 µM ginsenoside CK or CCCP plus 20 nM Rapamycin for 12 h. Subsequently, Tom20 antibody (Cell Signaling Technology, 42406, 1:100) was used to label Tom20 according to the immuno uorescence method described above. Finally, the cells were observed by uorescence confocal microscope (Olympus, BX51/DP71).
To observe the co-localization of Parkin and Tom20, GFP-Parkin plasmids were transfected into C2C12 cells by Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scienti c, L3000015). Then the cells were treated with 20 µM carbonyl cyanide-interchlorophenhydrazone (CCCP), CCCP plus 7.5 µM ginsenoside CK or CCCP plus 20 nM Rapamycin for 12 h. Subsequently, Tom20 antibody (Cell Signaling Technology, 42406, 1:100) was used to label Tom20 according to the immuno uorescence method described above. Finally, the cells were observed by uorescence confocal microscope (Olympus, BX51/DP71).
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