Proteins from the mitochondria of lung tissue were extracted as per the manufacturer’s instructions using a mitochondrial protein extraction kit (Beyotime). The Bradford protein assay was performed to determine the protein levels of mitochondrial Fis1, Drp1, Mfn2, Mfn1, OPA1, and β-actin. Polyclonal antibodies such as Fis1 (dilution ratio, 1: 50), Drp1 (dilution ratio, 1: 50), Mfn2 (dilution ratio, 1: 100), Mfn1 (dilution ratio, 1: 100), OPA1 (dilution ratio, 1: 50), and β-actin (dilution ratio, 1: 1000) were incubated overnight at 4°C with phosphate-buffered saline and bovine serum albumin. Further secondary antibodies were incubated at room temperature for 90 minutes. The Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to quantify band density.
Mitochondrial protein extraction kit
Manufactured by Beyotime
Sourced in China
The Mitochondrial Protein Extraction Kit is a laboratory tool designed to isolate and extract mitochondrial proteins from cellular samples. This kit provides a streamlined process for the efficient separation and purification of mitochondrial proteins, enabling researchers to study their structure, function, and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (1 mentions)
Protein a g agarose bead, by Beyotime (1 mentions)
Gapdh, by Merck Group (1 mentions)
α actinin, by Merck Group (1 mentions)
Rictor, by Abcam (1 mentions)
Hsp90, by Abcam (1 mentions)
Connexin 43, by Abcam (1 mentions)
Rictor, by Santa Cruz Biotechnology (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Akt1 2 3, by Santa Cruz Biotechnology (1 mentions)
Western blot lysis buffer, by Beyotime (1 mentions)
Acetyl lysine, by Abcam (1 mentions)
4 protocols using mitochondrial protein extraction kit
1
Mitochondrial Protein Expression Analysis
2
Quantifying Cytochrome C in Brain Tissues
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Mitochondrial and cytoplasmic extracts from brain tissues were prepared using mitochondrial protein extraction kit (Beyotime, China). The levels of cytochrome c in cytosolic and mitochondrial fractions were measured by the Quantikine rat cytochrome c immunoassay assay kit (R&D Systems, USA) according to the manufacturer's protocol. Data were expressed as ng/mg protein.
3
Mitochondrial Protein Extraction and Co-Immunoprecipitation
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Cell lysates were prepared in Western blot lysis buffer (Beyotime, Shanghai China). Mitochondrial/cytoplasmic proteins were obtained from samples with a mitochondrial protein extraction kit (Beyotime). Samples were coimmunoprecipitated using Protein A + G agarose beads (Beyotime) according to the manufacturer’s instructions. Western blotting was performed as previously reported [26 (link), 27 (link)]. Antibodies against thefollowing proteins were used: GAPDH, α-actinin (Sigma Aldrich), Akt1/2/3 (Ser 473), Akt1/2/3, Rictor, VDAC1 (Santa Cruz, TX, USA), cytochrome c, Oct4, mTOR, p-mTOR (Ser2481), HDAC6, TOM20 (Cell Signalling Technology, MA, USA), c-TNT, Connexin 43, Rictor, Hsp90, SIN1, G protein beta subunit like, and acetyl-lysine (Abcam, MA, USA). The membranes were incubated with horseradish peroxidase (HRP)-conjugated antibodies (Lianke, Hangzhou, China). All data analyses were carried out by using ImageJ software.
4
Mitochondrial Thioredoxin 2 Activity Assay
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Mitochondrial thioredoxin 2 activity was determined with an insulin disulfide reduction assay as reported previously [16 (link)]. Mitochondrial protein was extracted with a mitochondrial protein extraction kit (Beyotime Biotechnology, China), and Trx2 activity is presented as the OD412 fold change compared to the control on the basis of the absorption measurement obtained at a wavelength of 412 nm.
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