Purified proteins were mixed with NativePAGE sample buffer (ThermoFisher) and loaded into a NativePAGE 4–12% Bis-Tris gel (Thermo Fisher) according to the manufacturer’s instruction. The BN-PAGE gels were run for 2h at 150V and stained with Coomassie blue.
Nativepage 4 12 bis tris gel
Manufactured by Thermo Fisher Scientific
The NativePAGE 4–12% Bis-Tris gel is a pre-cast polyacrylamide gel designed for the separation and analysis of native proteins. It provides a gradient of pore sizes to enable the separation of a wide range of protein complexes under native conditions.
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4 protocols using nativepage 4 12 bis tris gel
1
Native PAGE Protein Analysis
2
Native PAGE Analysis of Purified Proteins
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Purified proteins were mixed with NativePAGE Sample Buffer (Thermo Fisher) and loaded into a NativePAGE 4%–12% Bis-Tris Gel (Thermo Fisher) according to the manufacturer’s instructions. The BN-PAGE gels were run for 2 h at 150 V and stained with Coomassie blue.
3
Mitochondrial Protein Complexes Characterization
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Isolated mitochondria were solubilized in 1X NativePage Sample Buffer supplemented with 1% Digitonin (Life Technologies). Samples were incubated on ice for 10min and then centrifuged at 20,000g for 30min at 4°C. Supernatants were isolated and total extracted protein quantified using the DC Protein Assay (Bio-Rad Laboratories). Samples were prepared and run on NativePage 4–12% Bis-Tris gels as per the manufacturer’s instructions (Life Technologies). For immunoblotting, samples were transferred onto PVDF membranes using Mini Trans-Bolt Cell (Bio-Rad Laboratories). Subsequent probing and imaging was performed as described above for immunoblotting. Loading was visualised using Coomassie Blue on a duplicate gel. In-gel assays were performed for complex I and II activity as described in 20 (link).
4
Isolation and Analysis of Mitochondrial Complexes
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Isolated mitochondria were solubilized in 1× NativePage Sample Buffer supplemented with 1% digitonin (Life Technologies). Samples were incubated on ice for 10 min and then centrifuged at 20,000×g for 30 min at 4 °C. Supernatants were isolated and total extracted protein was quantified using the DC Protein Assay (Bio-Rad Laboratories). Samples were prepared and run on NativePage 4–12% Bis-Tris gels as per the manufacturer’s instructions (Life Technologies). For immunoblotting, samples were transferred onto PVDF membranes using a Mini Trans-Bolt Cell (Bio-Rad Laboratories). Subsequent probing and imaging was performed as described above for immunoblotting. Loading was visualized using Coomassie Blue on a duplicate gel.
In-gel assays were performed for complex I and II activity as previously described35 (link).
In-gel assays were performed for complex I and II activity as previously described35 (link).
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