Total cell lysates were prepared and western blotting was performed as described previously [31 (link)]. Proteins (20–40 μg) were separated by SDS-PAGE on 4–10% resolving polyacrylamide gradient gels (Bio-Rad, USA) and electroblotted to PVDF membranes. The membranes were incubated with specific primary antibodies, followed by 1 hr incubation with appropriate peroxidase-coupled secondary antibodies. Signals were detected using a SuperSignal chemiluminescence kit (Thermo Fisher, USA) and images were captured using ImageLab software (Bio-Rad, USA). Band intensity was quantified using the Image J software. Separate aliquots were probed for β-actin to assess loading. Protein levels were expressed as relative expression/β-actin (mean± SD).
Resolving polyacrylamide gradient gels
Manufactured by Bio-Rad
Sourced in United States
4–10% resolving polyacrylamide gradient gels are laboratory equipment used for the separation and analysis of proteins based on their molecular weight. These gels feature a continuous gradient of acrylamide concentration, allowing for the effective separation of a wide range of protein sizes in a single electrophoresis run.
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2 protocols using resolving polyacrylamide gradient gels
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Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Levels
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Total cell lysates were prepared and western blotting was performed as described previously 31 . Proteins (20–40 μg) were separated by SDS–PAGE on 4–10% resolving polyacrylamide gradient gels (Bio‐Rad, Hercules, CA) and electroblotted to PVDF membranes. The membranes were incubated with specific primary antibodies, followed by 1 h incubation with appropriate peroxidase‐coupled secondary antibodies. Signals were detected using a SuperSignal chemiluminescence kit (Thermo Fisher, Rocksord, IL) and images were captured using ImageLab software (Bio‐Rad). Band intensity was quantified using the Image J software. Separate aliquots were probed for β‐actin to assess loading. Protein levels were expressed as relative expression/ β‐actin (mean ± SD).
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