Glomeruli were isolated by Dynabead perfusion. Cells were harvested by trypsinization and lysis was performed using RIPA buffer. Protein concentration was determined by BCA assay (Pierce Biotechnology, 23225). Equal amounts of protein were separated on SDS-PAGE. Afterward, gels were transferred to a polyvinyl difluoride membrane (iBlot Gel Transfer Stacks, Life Technologies, IB401001) by dry blotting technique (iBlot® 2 Dry Blotting System, Life technologies). Membranes were blocked in 5% PBS-BSA Following antibodies were used for western blot: anti β-ACTIN (Sigma, A9357), anti-alpha TUBULIN (Sigma, T6199), anti DRP-1 (Cell Signaling Technology, D6C7), anti TFAM (Abcam, ab131607), anti PGC-1α (Merck Milipore, 4C1.3), total OXPHOS Rodent WB Antibody Cocktail (Abcam, ab110413). Densitometry was performed using LabImage ID software (Kapelan Bio-Imaging).
Anti tfam
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-TFAM is a laboratory reagent that can be used to detect and quantify the levels of the TFAM protein in biological samples. TFAM (Transcription Factor A, Mitochondrial) is a key regulator of mitochondrial transcription and plays an important role in mitochondrial DNA maintenance and biogenesis. Anti-TFAM is a specific antibody that can be used in various analytical techniques, such as Western blotting, immunohistochemistry, or ELISA, to measure TFAM expression or localization.
Automatically generated - may contain errors
Lab products found in correlation
Anti nrf1, by Abcam (4 mentions)
Anti pgc 1α, by Abcam (3 mentions)
Anti p ampk, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti pgc 1α, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Mounting medium, by Thermo Fisher Scientific (2 mentions)
Anti tom20, by Santa Cruz Biotechnology (2 mentions)
Axiovert inverted fluorescence microscope, by Zeiss (2 mentions)
Anti pgc 1α antibody, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti ampk, by Cell Signaling Technology (2 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer stack, by Thermo Fisher Scientific (1 mentions)
Total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti drp1, by Cell Signaling Technology (1 mentions)
Hrp coupled secondary antibody, by Proteintech (1 mentions)
Pparγ, by Abcam (1 mentions)
18 protocols using anti tfam
1
Quantifying Mitochondrial Dynamics in Glomeruli
2
Western Blot Analysis of Cellular Signaling
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Total proteins from cells or tissue samples were lyzed in a lysis buffer and quantified using a BCA assay. Ten-cell lysates were loaded into each well in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 5% nonfat milk in tris-buffered saline and polysorbate 20 (TBST) for 2 h at room temperature and incubated with the indicated primary antibodies, including anti-AMPK-α, anti-p-AMPK-α, anti-PGC-1α, anti-TFAM, anti-LC3B, anti-ULK1, anti-PINK1, anti-Parkin, anti-mammalian target of rapamycin (mTOR), anti-sirtuin 1 (SIRT1), anti-peroxisome proliferator-activated receptor (PPAR)γ (Abcam, Cambridge, United Kingdom), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China) at 4°C overnight. After incubation with the HRP-coupled secondary antibody (Proteintech, Wuhan, China) at room temperature for 2 h, signals were detected using a super-enhanced chemiluminescence plus reagent and scanned and quantified by a ChemiDoc MP imaging system (Bio-Rad, CA, USA).
3
Western Blot Analysis of Myocardium Proteins
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Myocardium tissue was harvested for Western blot as described previously [23 (link)]. Cells of each group were harvested at appropriate time. Cells were washed three times with PBS and collected after ice-cold lysis buffer digestion. Protein lysates were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose (NC) membrane. Membranes were blocked with 5% milk in 1 × TBS-Tween-20 buffer and incubated overnight at 4°C with primary antibodies. Then, membranes were washed in Tris-buffered saline with Tween, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were developed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) and visualized with UVP Bio-Imaging Systems. Blot densities were analyzed using ImageJ Software (National Institutes of Health, Bethesda, MD).
Primary antibodies are the following: anti-SIRT1, anti-IRS2, anti-P-Akt S473, anti-P-Akt T308, anti-t-Akt, anti-acetylated protein, anti-PGC-1α, anti-NRF1, anti-NRF2, anti-ERR-α, anti-TFAM, anti-GAPDH, and anti-β-actin (all from Abcam, Cambridge, MA, USA). Secondary antibodies are the following: horseradish peroxidase-conjugated goat anti-rabbit and goat anti-rat (from Zhongshan Biotechnology Co. Ltd., Beijing, China).
Primary antibodies are the following: anti-SIRT1, anti-IRS2, anti-P-Akt S473, anti-P-Akt T308, anti-t-Akt, anti-acetylated protein, anti-PGC-1α, anti-NRF1, anti-NRF2, anti-ERR-α, anti-TFAM, anti-GAPDH, and anti-β-actin (all from Abcam, Cambridge, MA, USA). Secondary antibodies are the following: horseradish peroxidase-conjugated goat anti-rabbit and goat anti-rat (from Zhongshan Biotechnology Co. Ltd., Beijing, China).
4
Cytochrome C and Mitochondrial Protein Analysis
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HepG2 treated cells were harvested in RIPA buffer (50 mM Tris pH8.0, 150 mM NaCl, 0.1%SDS, 0.02% Sodium azide, 1% NP40, 0.5% Sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktails (Sigma–Aldrich, USA). Protein was quantified using BCA protein assay kit (Thermo Fisher Scientific, USA). SDS-PAGE was performed using 40 μg of protein sample on a 10% denaturing SDS gel and transferred on polyvinylidene fluoride membrane (Bio-Rad, California, USA). Each blot was incubated with specific anti-Cytochrome C (Abcam, USA) (dilution 1:1000), anti-TFAM (Abcam USA) (dilution 1:1000), anti-COX IV (Santacruz Biotechnology, USA) (1:200), and anti-tubulin (Sigma Aldrich, India) (dilution 1:1000) followed by a secondary. The blot images were captured using ChemiDoc™ XRS (GE Amersham Imager, USA). The blot intensity was quantified using Image J software and the results were reported relative to tubulin band density used as a load control [28 (link)].
5
Mitochondrial Protein Isolation and Analysis
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Liver mitochondria were isolated in a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris–HCl, 1 mM EDTA, and 5 mM EGTA, pH 7.4, at 4 °C according to [16 (link)]. In addition, 10 μg of mitochondrial proteins were used for Western immunoblotting analysis. Anti-TFAM (1:50,000), anti-VDAC (1:50,000, Abcam, Cambridge, UK), anti-OGG1 (1:2500, Abcam, Cambridge, UK), anti-APE1 (1:5000, Abcam, Cambridge, UK), anti-MFN2 (1:5000, Abnova, Taipei, Taiwan), anti-DRP1 (1:2500, Abnova, Taipei, Taiwan), anti-Cyt c (1:500, Pharmingen, San Diego, CA, USA), and anti-Lon Protease (1:10,000) were used as primary antibodies. The antibodies against TFAM and Lon were custom-made and kindly donated, respectively, by Dr. H. Hinagaki (Department of Chemistry, National Industrial Research Institute of Nagoya, Nagoya-shi, Aichi, Japan) and Dr. C. Suzuki (Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA). The proteins were detected by chemiluminescence and immunoreactive bands were quantified using the Image Lab Software (BioRad Laboratories Inc., Hercules, CA, USA) and normalized against VDAC-expression.
6
Immunohistochemical Analysis of Liver Tissue
7
Quantifying Mitochondrial Nucleoids in Hematopoietic Cells
8
Kidney Protein Extraction and Western Blot Analysis
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Whole kidney protein was extracted with lysis buffer. After centrifugation (13,000 rpm, 4 °C, 15 min), the lysate was mixed with 5× sample buffer and heated at 95 °C for 6 min. Total protein concentrations were measured using Bradford methods (BioRad Laboratories, Hercules, CA, USA). The lysate was subjected to SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were incubated overnight at 4 °C with the primary antibodies: anti-p-Src (1:1000; Cell Signaling Technology), anti-p-Fyn (1:1000; Santa Cruz Biotechnology), anti-PGC-1α (1:1000; Abcam), anti-TFAM (1:1000; Abcam), anti-NRF1 (1:1000; Abcam), anti-Cox4i1 (1:1000; Cusabio Biotech Co., Baltimore, MD, USA), anti-β-actin (1:1000), and anti-heat shock 70 kDa protein 8 (HSC70, 1:1000; Santa Cruz Biotechnology). The blots were reacted with peroxidase-conjugated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA, USA), followed by an enhanced chemiluminescent sensitive plus reaction (BioFX Laboratories, Inc., Owings Mills, MD, USA). The positive immunoreactive protein bands were detected by LAS-3000 film (FUJIFILM Corporation, Tokyo, Japan). Each blot density was normalized to β-actin or heat shock 70 kDa protein 8 and compared with that of each control.
9
Protein Expression Analysis in Skeletal Muscle
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Skeletal muscle tissues and L6 cells were lysed in RIPA lysis buffer and centrifuged (12 000 × g, 10 min, 4 °C). The lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membrane, immunoblotted with following primary antibodies of rabbit monoclonal anti-Sirt1, anti-Sirt3, anti-PGC-1α (BBI, Shanghai, China), anti-TFAM, anti-NRF1 and anti-GAPDH (ABCAM, Cambridge, UK), and then incubated with secondary goat anti-rabbit antibody (Cell Signaling, USA). The bands were visualized with enhanced chemiluminescence solution (ECL, Biotanon) and detected by Image Lab camera (Bio-rad, Germany), and then quantified by densitometry scanning with ImageJ software.
10
Evaluation of Mitochondrial Regulators in Cells
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Western blot was conducted as previously described [4 (link), 5 (link)]. Briefly, proteins were extracted from cells and cochlea tissues using radioimmunoprecipitation assay lysis buffer (Thermo plus, USA). Protein samples (20 μg) were resolved on 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). After blocking with 5% nonfat milk, the membranes were incubated with anti-PGC-1α (1 : 1000, Abcam, USA), anti-TFAM (1 : 1000, Abcam), anti-p-AMPK (1 : 1000, Cell Signaling Technology, USA), anti-AMPK (1 : 1000, Cell Signaling Technology, USA), and anti-Arid5b (1 : 500, Abgent, USA) at 4°C overnight, followed with secondary antibodies (1 : 10000) at room temperature for 1 h. Then, the immunoreactive bands were detected using enhanced chemiluminescence (Millipore, USA). Band intensities were analyzed using NIH ImageJ. β-Actin was applied as loading and internal control.
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