A plant mitochondrial isolation kit (catalogue number P0045, Biohao, Wuhan, China) was used to isolate crude mitochondria from WT and dek55–1 seed tissue, excluding the pericarp (at 15 DAP), for BN-PAGE and complex I activity analysis. The collected mitochondrial precipitate was redissolved in 35 μL of solution buffer (50 mmol/L bis-Tris, 6 N HCl, 50 mmol/L NaCl, 10% w/v glycerol, 0.001% Ponceau S; pH 7.2) containing 20% n-dodecyl-b-D-maltoside (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 1%) and then kept on ice for 30 min. The suspension was then centrifuged at 4 °C, after which the supernatant was collected and loaded on preprepared gradient gels (BN1002BOX, Thermo Fisher Scientific), and electrophoresis was performed according to the manufacturer’s instructions. Afterwards, the gels were placed in 100 mL of fixing solution (methanol:ddH2O:acetic acid, 4:5:1) for 30 min and then transferred to 0.02% Coomassie R-250 stain (Sigma-Aldrich, St. Louis, MO, USA) for mitochondrial complex abundance analysis. The gel strips were incubated in assay buffer (25 mg nitro blue tetrazolium and 100 μL of NADH (10 mg/mL) combined with 10 mL of 5 mmol/L Tris/HCl; pH 7.4) (Sigma-Aldrich) for 5 min, and the reaction was terminated with the fixing solution (40% methanol:10% acetic acid (v/v)) for analysis of complex I activity [44 (link)].
Coomassie r 250 stain
Manufactured by Merck Group
Sourced in United States
Coomassie R-250 stain is a protein-binding dye used for the detection and quantification of proteins in various laboratory applications. It is a blue dye that binds to proteins, resulting in a blue-colored complex that can be visualized and measured.
Automatically generated - may contain errors
2 protocols using coomassie r 250 stain
1
Mitochondrial Isolation and Analysis
2
Mitochondrial Isolation and Complex I Analysis
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A mitochondrial isolation kit (catalogue number P0045, Biohao, Wuhan, China) was used to isolate crude mitochondria from WT and dek55-1 seed tissue, excluding the pericarp (at 15 DAP), for BN-PAGE and complex I activity analysis. The collected mitochondrial precipitate was redissolved in 35 μL of solution buffer (50 mmol/L bis-Tris, 6 N HCl, 50 mmol/L NaCl, 10% w/v glycerol, 0.001% Ponceau S; pH 7.2) containing 20% n-dodecyl-b-D-maltoside (Sigma-Aldrich, St. Louis, MO, USA) to a nal concentration of 1%) and then kept on ice for 30 min. The suspension was then centrifuged at 4°C, after which the supernatant was collected and loaded on preprepared gradient gels (BN1002BOX, Thermo Fisher Scienti c), and electrophoresis was performed according to the manufacturer's instructions. Afterwards, the gels were placed in 100 mL of xing solution (methanol:ddH 2 O:acetic acid, 4:5:1) for 30 min and then transferred to 0.02% Coomassie R-250 stain (Sigma-Aldrich, St. Louis, MO, USA) for mitochondrial complex abundance analysis. The gel strips were incubated in assay buffer (25 mg nitro blue tetrazolium and 100 μL of NADH (10 mg/mL) combined with 10 mL of 5 mmol/L Tris/HCl; pH 7.4) (Sigma-Aldrich) for 5 min, and the reaction was terminated with the xing solution (40% methanol:10% acetic acid (v/v)) for analysis of complex I activity [44] .
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