For Blue-Native electrophoresis, proteins of isolated mitochondrial fractions (2 mg protein for immunoblotting with anti-F1b and 20 mg for immunoblotting with anti-Cyt1 antibodies) were incubated in 100 ml of Blue-Native PAGE buffer (50 mM NaCl, 50 mM imidazole, 1 mM EDTA, 3% digitonin, pH 7.0) for 10 min at 4 C (Wittig et al., 2006) and mixed with 13 ml of Native PAGE 5% G-250 Sample Additive (Invitrogen) and 18 ml of 30% glycerol. The samples were separated by electrophoresis on NativePAGE 3%-12% Bis-Tris gels (Invitrogen) with NativePAGE Anode Buffer and NativePAGE Dark Blue Cathode Buffer (Invitrogen) for 35 min at 150 V and then with NativePAGE Anode Buffer and NativePAGE Light Blue Cathode Buffer (Invitrogen) for 140 min at 250 V.
Nativepage anode buffer
Manufactured by Thermo Fisher Scientific
The NativePAGE Anode Buffer is a specialized buffer solution designed for use in native polyacrylamide gel electrophoresis (native PAGE) applications. It is formulated to maintain the native, non-denaturing conditions of proteins during the electrophoresis process, allowing for the analysis of protein structure and function.
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Lab products found in correlation
Nativepage system, by Thermo Fisher Scientific (1 mentions)
Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions)
Dark blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Nativepage 3 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nativepage 5 g 250 sample additive, by Thermo Fisher Scientific (1 mentions)
2 protocols using nativepage anode buffer
1
Mitochondrial Protein Complex Separation by Blue-Native Electrophoresis
2
Blue Native PAGE for Mitochondrial Complexes
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Crude mitochondrial fractions were isolated and normalized for total protein content as described above. Blue Native (BN) PAGE was performed using the Invitrogen NativePAGE system. 100 µg of mitochondria were pelleted at 10,000 x g for 10 min at 4°C and resuspended in 1x pink lysis buffer (Invitrogen, BN20032). Digitonin (GoldBio D-180–2.5) was added to a final concentration of 1% mass/volume. Samples were incubated on ice for 15 min, then spun for 20 min at 20,000 x g. 6 µL of NativePAGE sample buffer (Invitrogen, BN20041) was added and 10 µL of sample was run on precast 3–12% NativePAGE gels (Invitrogen, BN2011B × 10) with NativePAGE anode buffer (Invitrogen, BN2001) and dark blue cathode buffer (Invitrogen, BN2002) at 150 V for 1 hr then switched to light blue cathode buffer (Invitrogen, BN2002) and run at 30 V overnight. Gels were subsequently transferred to PVDF at 100 V, washed with methanol, and blotted with the indicated primary antibodies which are listed in the key resources table according to the manufacturers’ recommendations. Secondary anti-mouse HRP antibody listed in the key resources table and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo, 34096) was used to visualize bands on film (GeneMate, F-9024−8 × 10).
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