Mitochondrial extraction from fresh rat brain tissue performed using a kit (C3606, Beyotime) (Mao et al., 2019). First, 100 mg tissue was cut under an ice bath and placed in a 1.5 mL centrifuge tube, followed by washing with PBS. The brain tissue block was cut into very small tissue fragments with scissors. Pre-cooled mitochondrial separation reagent A (10 μL/mg) was added and the mixture was homogenized in an ice bath. Low-temperature centrifugation was performed at 1000 × g and 4°C for 5 minutes. The supernatant was transferred to another centrifuge tube and centrifuged at 3500 × g and 4°C for 10 minutes. The supernatant was carefully removed the precipitates contained the isolated mitochondria.
C3606
Manufactured by Beyotime
Sourced in China
The C3606 is a laboratory equipment designed for measuring the electrical conductivity of solutions. It features a digital display and allows for precise measurement of conductivity values.
Automatically generated - may contain errors
Lab products found in correlation
C3601, by Beyotime (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
Ab13533, by Abcam (1 mentions)
Ab137037, by Abcam (1 mentions)
Affinipure goat anti mouse igg, by Proteintech (1 mentions)
Af0863, by Affinity Biosciences (1 mentions)
Bc3710, by Solarbio (1 mentions)
R0050, by Solarbio (1 mentions)
Df7198, by Affinity Biosciences (1 mentions)
Bf0591, by Affinity Biosciences (1 mentions)
Af5135, by Affinity Biosciences (1 mentions)
Bca protein quantitative kit, by Beyotime (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Albumin bovine 5, by Solarbio (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
8 protocols using c3606
1
Mitochondrial Extraction from Rat Brain
2
Isolation of Muscle Mitochondria
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3
Protein Extraction and Western Blotting for Subcellular Analysis
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We used the same method as before to perform Western blotting.37 Before Western blotting, we used a total protein extraction kit (BC3710, Solarbio), cellular mitochondrial isolation kit (C3601, Beyotime) or tissue mitochondrial isolation kit (C3606, Beyotime), and nuclear protein extraction kit (R0050, Solarbio) to extract total protein, mitochondrial protein or de‐mitochondrial cytoplasmic protein, and nuclear protein, respectively. The primary antibodies used were anti‐PARP‐1 (1:1000, DF7198, Affinity); anti‐AIF (1:1000, BF0591, Affinity); anti‐COX IV (1:1000, AF5468, Affinity); anti‐Histone H3 (1:1000, AF0863, Affinity); anti‐β‐actin (1:10,000, 66009‐1‐Ig, Protentech); anti‐LC3BI/II (1:1000, A5402, Affinity); anti‐PINK1 (1:200, PA5‐85930, Invitrogen); anti‐Parkin (1:200, 702785, Invitrogen); anti‐SIRT3 (1:1000, Affinity, AF5135); anti‐SOD2/MnSOD (acetyl K68) (1:5000, ab137037, Abcam); and anti‐SOD2/MnSOD (1:5000, ab13533, Abcam). The second antibodies used were HRP‐conjugated AffiniPure Goat Anti‐Mouse IgG (1:10,000, Proteintech) and HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG (1:10,000, Proteintech). Finally, PVDF molds were visualized on a Tanon 2500R gel imaging system (Tanon) using a developer (Tanon), and the band intensity was quantified using ImageJ 1.39V software.
4
Protein Extraction and Western Blot Analysis
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Total proteins were extracted by incubating the cell pellet or kidney in RIPA buffer (P0013D, Beyotime) supplemented with 1% PMSF and 1% phosphatase inhibitors. Protein concentration was measured using a BCA protein quantitative kit (P0011, Beyotime). For the extraction of mitochondrial proteins, the mitochondria were first isolated from the cell pellet or kidney tissues after incubation with mitochondrial separation reagent (C3606, C3601, Beyotime), followed by grinding with a glass homogenizer. Isolated mitochondria were resuspended in RIPA buffer for protein extraction.
Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% albumin bovine V (Solarbio A8020) at room temperature for 1 h, the membrane was incubated with the primary antibodies (Table S2 ) at 4 °C overnight. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibodies at room temperature for 1 h, and exposed to ECL reagent (WBKLS0500, Millipore).
Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% albumin bovine V (Solarbio A8020) at room temperature for 1 h, the membrane was incubated with the primary antibodies (
5
Mitochondrial Membrane Potential Assay
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Mitochondria were extracted from renal tissue using a mitochondrial extraction kit (C3606, Beyotime, China). Briefly, fresh kidneys were harvested, cut into small pieces, and washed thrice with precooled PBS. After digestion with trypsin, the renal tissues were homogenized in mitochondrial isolation reagent using a Dounce tissue grinder on ice. The homogenate was then centrifuged at 600 g for 10 min, and the supernatant was centrifuged at 1,500 g for 15 min to isolate the mitochondria. The change in MMP was measured by the JC-1 fluorescent probe, and the JC-1 red/green fluorescence intensity ratio was used to represent MMP. Fresh isolated mitochondria were incubated with 10 μg/ml JC-1 (at 37°C for 20 min), and the fluorescence intensity was measured by a Synergy H1 fluorometer microplate reader (BioTek, United States).
6
Mitochondrial and Cytosolic Fractionation
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Mitochondrial and cytosolic fractions were isolated using a commercially available cytosol/mitochondria fractionation kit according to the manufacturer’s protocol (Beyotime, C3606).
7
Mitochondrial Isolation from Adipose Tissue
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Brown adipose tissue or mature adipocytes were collected, and mitochondrial fractions were extracted using a mitochondrial isolation kit according to the manufacturer’s instructions (Beyotime, C3606, China). Protein was quantified using the bicinchoninic acid method.
8
Hippocampus Mitochondrial Membrane Potential
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Mitochondria of hippocampus tissue in rats was extracted and purified by differential centrifugation according to the manufacturer's directions (C3606 Beyotime, Shanghai, China). The MMP of hippocampus tissue was detected by JC-1 fluorescent probe staining method and the experiments were repeated three times. According to the instructions of the kit (C2006 Beyotime, Shanghai, China), the fluorescence was measured using EnSpire multimode plate reader (Perkin Elmer, MA, USA) at 485 nm excitation/590 nm emission.
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