Protein was extracted from left ventricle tissues using 0.2 ml precooled lysis buffer/20 mg tissue. Protein concentration was measured by BCA protein concentration assay kit (Beyotime, Shanghai, China), and the supernatant was denatured at 95°C for 5 min in Laemmli sample buffer. Samples were subjected to 8% or 10% SDS-PAGE gels. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck, U.S.A.), which was incubated in the blocking buffer (5% non-fat milk, 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, 0.01% Tween 20) for 1 h at room temperature and was followed by incubation with anti-COX1 (ab203912, Abcam, U.K.), anti-ATPase6 (A8193, Abclonal, China), anti-SIRT1 (sc-74465, Santa Cruz, U.S.A.), anti-PGC-1α (sc-518025, Santa Cruz, U.S.A.), anti-NRF1 (ab175932, Abcam, U.K.) and anti-TFAM (sc-166965, Santa Cruz, U.S.A.) overnight at 4°C. Binding of the primary antibody was detected by an enhanced chemiluminescence method (BeyoECL Star, P0018A, Beyotime) using horseradish peroxidase-conjugated secondary antibodies (Beyotime, Shanghai, China). The quantification of protein expression was performed using ImageJ.
Ab203912
Manufactured by Abcam
Sourced in United Kingdom
Ab203912 is a lab equipment product offered by Abcam. It is a device designed to facilitate specific laboratory tasks. The core function of this product is to enable researchers to perform their experiments effectively. No additional information can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab175932, by Abcam (2 mentions)
Ab14705, by Abcam (2 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Sc 166965, by Santa Cruz Biotechnology (1 mentions)
Sc 518025, by Santa Cruz Biotechnology (1 mentions)
Sc 74465, by Santa Cruz Biotechnology (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab252432, by Abcam (1 mentions)
Ab106814, by Abcam (1 mentions)
Ab280888, by Abcam (1 mentions)
Ab40855, by Abcam (1 mentions)
Cycloheximide c7698, by Merck Group (1 mentions)
Pa5 109993, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit immunoglobulin g, by Jackson ImmunoResearch (1 mentions)
Ab10983, by Abcam (1 mentions)
Ab186734, by Abcam (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
5 protocols using ab203912
1
Protein Expression Analysis in Cardiac Tissue
2
Astragaloside IV Antioxidant Mechanism
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Astragaloside IV (C41H68O14, A800922, purity ≥98.5%) was purchased from Maclin Inc. and the chemical structure is shown as Figure 1A . ROS fluorescent probe‐DHE (R001, Vigorous Biotechnology) used for detection of ROS in tissues and DCFH‐DA (KM0062) used for detection of cellular ROS was purchased from Beijing Biolab Technology Co. The antibodies used in this study including NRF1 (ab175932, abcam), PGC‐1α (ab106814, abcam), TFAM (ab252432, abcam), Caspase3 (9662, CST), cleaved‐caspase3 (ab32042, abcam), Bax (ab111391, abcam), Bcl2 (2872, CST), α‐SMA (A5228, sigma), p‐smad2 (ab280888, abcam), smad2 (ab40855, abcam), MT‐CO1 (ab203912, abcam), MT‐ND6 (PA5‐109993, Invitrogen) and MT‐ATP6 (PA5‐116789, Invitrogen). MG‐132 (M7749) and Cycloheximide (C7698) were purchased from Sigma.
3
Mitochondrial Protein Analysis by Western Blot
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Whole-cell lysates and mitochondrial lysates were prepared in lysis buffer (10 mM Tris–HCl, pH 8, 140 mM NaCl, 5 mM EDTA, 0.025% NaN3, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 1 × Complete Protease Inhibitor Cocktail (Santa Cruz, sc-29131)). The lysates (25–50 μg) were resolved by SDS-PAGE and proteins were transferred to PVDF membranes (Millipore). Membranes were incubated with the primary antibody, followed by incubation with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Jackson Immunoresearch, #111-035-003), and proteins were detected by enhanced chemiluminescence (GE). The primary antibodies for western blotting were anti-MTCO1 (COX I) antibody (Abcam, ab203912), anti-UCP1 antibody (Abcam, ab10983) and anti-Tomm20 antibody (Abcam, ab186734). These antibodies were used at a dilution of 1:1000.
4
Mitochondrial Protein Profiling in Tissue Samples
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Powdered tissue samples were homogenized in a RIPA lysis buffer (Cell Signaling, Danvers, MA,) with a protease inhibitor cocktail (Roche, Basel, Switzerland). Samples were centrifuged at 16,000 g for 20 minutes and supernatant was collected. Protein concentration was measured using a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) and 30 μg of protein was loaded onto a 4-20% SDS-PAGE gel (Bio-Rad) and run at 200 V for 45 minutes. The protein was transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) at 100 V for 30 minutes using the plate electrode. Total protein normalization was assayed using a Revert 700 Total Protein Stain Kit (LI-COR, Lincoln, NE, USA) Supplementary Figures 1 –4 . The membrane was blocked using a blocking buffer (LI-COR) then probed with primary antibody overnight at 4° C. Primary antibodies against MTCO1 (Complex IV) (ab14705,1:2000; ab203912, 1:1000), NDUFB-8 (Complex I) (ab110242, 1:2000), and Citrate Synthase (ab129095, 1:1000) were obtained from Abcam (Cambridge, United Kingdom). NIR fluorescent secondary antibodies (LI-COR) were applied and membranes were imaged using a LI-COR Odyssey imager.
5
Immunostaining Protocol for Neuronal and Fibroblast Cells
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For primary hippocampal neurons and NIH/3T3 cells, cells were rinsed with PBS and fixed with 4% PFA for 10 minutes. Cell were permeabilized with PBST (PBS with 0.2% Triton X-100) for 10 minutes and blocked with 1% BSA in PBST for 30 minutes. Primary antibodies were incubated at 4℃ for overnight. Antibodies against Tau1 (Millipore, MAB3420, monoclonal), FLAG (Sigma, F7425, polyclonal), and MTCO1 (Abcam, ab203912, monoclonal) were used for immunostaining. The cells were washed three times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen).
Then these cells were washed three times with PBS and coverslips were mounted on slide glasses.
Images were taken by using LSM780 confocal microscopy. For Neuro2A, cells were washed with icecold PBS followed by fixation using 4% PFA/sucrose for 15 minutes. After 3 times washing with PBS, cells were permeabilized with PBST (PBS with 0.5% Triton X-100) for 15 minutes and blocked with 1% BSA in PBS for 1 hour. Primary antibodies were incubated at 4℃ for overnight. Cells were washed 3 times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen) for 1 hour at room temperature. Cells were washed three times with PBS and coverslips were mounted on slide glasses.
Antibodies against GFP (Abcam, ab 6556, polyclonal) and MT-CO1 (Abcam, ab14705, monoclonal) were used for immunostaining.
Then these cells were washed three times with PBS and coverslips were mounted on slide glasses.
Images were taken by using LSM780 confocal microscopy. For Neuro2A, cells were washed with icecold PBS followed by fixation using 4% PFA/sucrose for 15 minutes. After 3 times washing with PBS, cells were permeabilized with PBST (PBS with 0.5% Triton X-100) for 15 minutes and blocked with 1% BSA in PBS for 1 hour. Primary antibodies were incubated at 4℃ for overnight. Cells were washed 3 times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen) for 1 hour at room temperature. Cells were washed three times with PBS and coverslips were mounted on slide glasses.
Antibodies against GFP (Abcam, ab 6556, polyclonal) and MT-CO1 (Abcam, ab14705, monoclonal) were used for immunostaining.
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