Novex 4 16 bis tris gels

Mitochondria for blue native PAGE (BN-PAGE) and complex activity analysis were isolated from kernels without pericarp of self-pollinated segregating ears at 15 DAP as described previously (Chen et al., 2017 (link)). The enriched mitochondria were resuspended in 40 μl of sample buffer [50 mM Bis-Tris, 6 N HCl, 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S] with 20% n-dodecyl-β-d-maltoside added to a final concentration of 1%, and incubated for 30 min on ice. The samples were centrifuged at 20 000 g for 30 min at 4 °C and loaded on native PAGE Novex 4–16% Bis-Tris gels (Invitrogen, BN1002BOX). The electrophoresis was performed following the manufacturer’s protocol. The gels were stained with Coomassie Blue R-250 (Sigma-Aldrich). The activities of mitochondrial complexes were determined by colorimetry as described previously (Luo et al., 2008 ; Muhling et al., 2010 (link); Gadicherla et al., 2012 (link)).

The purified proteins were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS–PAGE) using 4–12% Bis‐Tris NuPAGE gels (Invitrogen). Prior to SDS–PAGE analysis, protein samples were heated in the presence of 10 mM DTT at 90°C for 10 min. For Western blot analysis, proteins from SDS–PAGE gels were transferred onto a nitrocellulose membrane using iBlot blotting system (Thermo Scientific). Membranes were blocked for 1 h in TBST (10 mM Tris–HCl, 150 mM NaCl, and 0.05% Tween 20, pH 8) containing 2% (w/v) biotin‐free bovine serum albumin (BSA). The Strep‐II‐tagged proteins were immunodetected using the alkaline phosphatase‐conjugated Strep‐Tactin (IBA) and the color reaction was developed with 5‐bromo‐4‐chloro‐3‐indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT). The Blue native PAGE was performed using the Novex 4–16% Bis‐Tris gels (Invitrogen) according to the manufacturer's instructions.