BN-PAGE was performed as described previously [13 (link)]. The proteins from SKM mitochondria were extracted with 5% digitonin (Invitrogen, Carlsbad, CA; protein:detergent ratio of 1:10) and 4 × buffer on ice for 30 min. After centrifugation at 10,000 g for 10 min at 4 °C, the supernatants were collected. The remaining lysate was combined with Coomassie blue G-250 dye (Invitrogen; protein:detergent ratio of 1:10) and added to 3%–12% NativePAGE Novex Bis-Tris Gel (Invitrogen), then separated by electrophoresis using Anode and Cathode buffer (Invitrogen) at 10 mA for 1 h and at 150 V for 2 h on ice. The protein complex in the samples after the electrophoresis was denatured by denaturing buffer (Tris [20 mmol], glycine [200 mmol], 1% SDS). The gels were then transferred by electroblotting to PVDF membranes (Bio-Rad) using transfer buffer at 25 V for 2 h.
Coomassie blue g 250 dye
Manufactured by Thermo Fisher Scientific
Coomassie blue G-250 dye is a protein-binding dye used in various laboratory applications. It is a blue-colored dye that binds to proteins, allowing for the quantification and visualization of protein samples.
Automatically generated - may contain errors
2 protocols using coomassie blue g 250 dye
1
Mitochondrial Protein Complex Isolation
2
Mitochondrial Protein Complexes Analysis
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BN-PAGE was performed as described previously [21 (link)]. Briefly, mitochondrial proteins were extracted with 5% digitonin (Invitrogen, Carlsbad, CA; protein:detergent ratio of 1:10) and 4× buffer (Invitrogen) on ice for 30 min. After centrifugation at 10,000 g for 10 min at 4 °C, the supernatants were collected. The remaining lysates were combined with Coomassie blue G-250 dye (Invitrogen; protein:detergent ratio of 1:10) and added to 3–12% NativePAGE Novex Bis-Tris Gel (Invitrogen), then separated by electrophoresis using Anode and Cathode buffer (Invitrogen) at 10 mA for 1 h and at 150 V for 2 h on ice. Protein complexes in the samples after electrophoresis were denatured using denaturing buffer (20 mmol Tris, 200 mmol glycine, and 1% SDS). The gels were then transferred by electroblotting to PVDF membranes (Bio-Rad) using transfer buffer at 25 V for 2 h.
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