Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was performed by a procedure adapted from the laboratory of Prof. Jörg Nickelsen, Ludwig Maximilians University of Munich [30] (link). Briefly, thylakoids corresponding to 500 µg protein were pelleted by centrifugation at 20,000×g for 20 min at 4°C and resuspended in 50 µL ACA buffer (50 mM Bis-Tris, pH 7.0, 750 mM aminocaproic acid, 0.5 mM EDTA). The thylakoids were solubilized by adding 7.5 µL 10% (w/v) β-DM and incubating on ice for 30 min. Samples were centrifuged at 20,000×g for 30 min at 4°C and supernatant was mixed with 8 µL loading buffer (750 mM aminocaproic acid, 5% (w/v) Coomassie G-250). 25 µL sample was loaded on a NativePAGE Novex 4–16% Bis-Tris Protein Gel (Life Technologies). Gel electrophoresis was performed at 4°C by first running at 40 V constant for 7 h with blue catode buffer (50 mM tricine, 15 mM Bis-Tris, pH 7.0, 0.2% (w/v) Coomassie G-250) and clear anode buffer (50 mM Bis-Tris, pH 7.0), then at 300 V constant in clear catode buffer (50 mM tricine, 15 mM Bis-Tris, pH 7.0) and clear anode buffer until reaching the end of the gel.
Nativepage novex 4 16 bis tris protein gel
Manufactured by Thermo Fisher Scientific
NativePAGE Novex 4–16% Bis-Tris Protein Gel is a pre-cast polyacrylamide gel used for native protein separation and analysis. It is designed to maintain the native structure and function of proteins during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Ab14713, by Abcam (2 mentions)
Ab110258, by Abcam (2 mentions)
Ab203832, by Abcam (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Nativepage sample prep kit, by Thermo Fisher Scientific (2 mentions)
Hrp linked donkey anti rabbit igg, by GE Healthcare (2 mentions)
Hrp linked sheep anti mouse igg, by GE Healthcare (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Western lightning plus ecl, by PerkinElmer (2 mentions)
3 protocols using nativepage novex 4 16 bis tris protein gel
1
Blue-native PAGE Analysis of Thylakoid Complexes
2
Native PAGE Analysis of Mitochondrial Complexes
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As previously described (Riessland et al., 2019 ), for the assessment of mitochondrial complexes, cell lysates were prepared using the NativePAGE Sample Prep Kit (Life Technologies) and solubilized with 1% digitonin. A BCA assay (Thermo Scientific) was used to determine the protein concentrations as described above. Equal amounts of protein were then loaded onto a NativePAGE Novex 4-16% Bis-Tris Protein Gel (Life Technologies). A wet blotting method was used to transfer the proteins onto a PVDF membrane (Life Technologies, 0.45 μm), the membrane was then incubated with 8% acetic acid for 15min and washed with methanol and water before being blocked with 5% BSA in 20 mM Tris, 150 mM NaCl, and 0.1% (w/v) Tween 20, pH 7.5, for 2 hours. The membrane was subsequently immunoblotted with the respective primary antibody at 4 °C overnight. The primary antibodies used were NDUFA9 (a CI Subunit) (1/2500 by vol; ab14713: Abcam), UQCRC2 (a CIII Subunit) (1/2500 by vol; ab203832: Abcam), or MT-CO2 (a CIV Subunit) (1/2500 by vol; ab110258: Abcam). Primary antibodies were detected using either HRP-linked donkey anti–rabbit IgG (GE Healthcare, #NA934V; 1:10,000) or HRP-linked sheep anti–mouse IgG (GE Healthcare, #NA931V, 1:10,000) together with Western Lightning Plus-ECL (Perkin Elmer, #NEL105001EA).
3
Mitochondrial Complex Assessment by Western Blot
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As previously described (Riessland et al., 2019 (link)), for the assessment of mitochondrial complexes, cell lysates were prepared using the NativePAGE Sample Prep Kit (Life Technologies) and solubilized with 1% digitonin. A BCA assay (Thermo Scientific) was used to determine the protein concentrations as described above. Equal amounts of protein were then loaded onto a NativePAGE Novex 4%–16% Bis‐Tris Protein Gel (Life Technologies). A wet blotting method was used to transfer the proteins onto a PVDF membrane (Life Technologies, 0.45 μm), the membrane was then incubated with 8% acetic acid for 15 min and washed with methanol and water before being blocked with 5% BSA in 20 mM Tris, 150 mM NaCl, and 0.1% (w/v) Tween 20, pH 7.5, for 2 h. The membrane was subsequently immunoblotted with the respective primary antibody at 4°C overnight. The primary antibodies used were NDUFA9 (a CI Subunit) (1/2500 by vol; ab14713: Abcam), UQCRC2 (a CIII Subunit) (1/2500 by vol; ab203832: Abcam), or MT‐CO2 (a CIV Subunit) (1/2500 by vol; ab110258: Abcam). Primary antibodies were detected using either HRP‐linked donkey anti–rabbit IgG (GE Healthcare, #NA934V; 1:10,000) or HRP‐linked sheep anti–mouse IgG (GE Healthcare, #NA931V, 1:10,000) together with Western Lightning Plus‐ECL (Perkin Elmer, #NEL105001EA).
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