At experimental endpoints, cells were washed twice with ice-cold DPBS and then lysed in 1% NP-40 lysis buffer supplemented with Halt Protease and phosphatase inhibitors for 15 min on ice. A Bio-Rad protein assay was used to normalize sample loading before SDS-PAGE. Samples were diluted in Laemmli sample loading buffer, heated for 5 min at 95°C, and then loaded onto precast 4 to 20% polyacrylamide tris-glycine gels (Bio-Rad). For CRLS1, SDHC, and SDHD IB, samples were instead heated for 30 min at 50°C to prevent precipitation. After SDS-PAGE, protein was transferred to a 0.45-μm nitrocellulose membrane by a semi-dry transfer system (Cytiva). Membranes were blocked with 5% BSA and 0.1% Tween 20 (IB blocking buffer) for 30 min at RT and then incubated with primary antibody in IB blocking buffer overnight at 4°C. Blots were developed using LI-COR IRdye secondary antibodies and an Odyssey IR Imager.Quantification of IBs was performed using ImageJ densitometric gel analysis protocol for 1D gels. A list of all antibody sources, concentrations, and applications is found in table S1.
4 to 20 polyacrylamide tris glycine gels
Manufactured by Bio-Rad
4 to 20% polyacrylamide tris-glycine gels are precast electrophoresis gels used for the separation and analysis of proteins based on their molecular weight. They feature a linear gradient of polyacrylamide concentrations ranging from 4% to 20%, allowing for effective separation of a wide range of protein sizes.
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2 protocols using 4 to 20 polyacrylamide tris glycine gels
1
Immunoblotting Protocol for Protein Detection
2
Immunoblotting Protocol for Protein Detection
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At experimental endpoints, cells were washed twice with ice-cold DPBS and then lysed in 1% NP-40 lysis buffer supplemented with Halt Protease and phosphatase inhibitors for 15 min on ice. A Bio-Rad protein assay was used to normalize sample loading before SDS-PAGE. Samples were diluted in Laemmli sample loading buffer, heated for 5 min at 95°C, and then loaded onto precast 4 to 20% polyacrylamide tris-glycine gels (Bio-Rad). For CRLS1, SDHC, and SDHD IB, samples were instead heated for 30 min at 50°C to prevent precipitation. After SDS-PAGE, protein was transferred to a 0.45-μm nitrocellulose membrane by a semi-dry transfer system (Cytiva). Membranes were blocked with 5% BSA and 0.1% Tween 20 (IB blocking buffer) for 30 min at RT and then incubated with primary antibody in IB blocking buffer overnight at 4°C. Blots were developed using LI-COR IRdye secondary antibodies and an Odyssey IR Imager.Quantification of IBs was performed using ImageJ densitometric gel analysis protocol for 1D gels. A list of all antibody sources, concentrations, and applications is found in table S1.
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