Treated cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in a minimum volume of 1X cell lysis buffer [1% Nonidet P-40; 50 mM Tris pH 7.4; 10 mM NaCl; 1 mM NaF; 5 mM MgCl2; 0.1 mM EDTA; 1 mM PMSF; and COMPLETE™ protease inhibitor cocktail tablet (Roche Diagnostocs Corp.)]. Protein content was determined using the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Cell extracts (10 μg/lane) were resolved by 4–12% gradient SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and electro-transferred onto nitrocellulose membranes. Following the transfer, membranes were stained with Ponceau S to confirm equal protein loading. Membranes were blocked with PBS/0.1% Tween-20 (PBST) and 10% skim milk and incubated with antibodies in PBST overnight at 4°C. Following incubation with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies, signals were detected using the SuperSignal Chemiluminescent Reagent (Pierce Chemical Co., Rockford, IL) with exposure of blots onto X-ray films.
Gradient sds polyacrylamide gel electrophoresis
Manufactured by Bio-Rad
4–12% gradient SDS-polyacrylamide gel electrophoresis is a laboratory technique used for the separation and analysis of proteins. It utilizes a gradient of polyacrylamide concentrations to create a porous gel matrix that separates proteins based on their molecular weight. The addition of sodium dodecyl sulfate (SDS) denatures the proteins, ensuring they migrate through the gel based on their size rather than their charge or shape.
Automatically generated - may contain errors
2 protocols using gradient sds polyacrylamide gel electrophoresis
1
Western Blotting for Protein Analysis
2
Western Blotting for Protein Analysis
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Treated cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in a minimum volume of 1X cell lysis buffer [1% Nonidet P-40; 50 mM Tris pH 7.4; 10 mM NaCl; 1 mM NaF; 5 mM MgCl2; 0.1 mM EDTA; 1 mM PMSF; and COMPLETE™ protease inhibitor cocktail tablet (Roche Diagnostocs Corp.)]. Protein content was determined using the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Cell extracts (10 μg/lane) were resolved by 4–12% gradient SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and electro-transferred onto nitrocellulose membranes. Following the transfer, membranes were stained with Ponceau S to confirm equal protein loading. Membranes were blocked with PBS/0.1% Tween-20 (PBST) and 10% skim milk and incubated with antibodies in PBST overnight at 4°C. Following incubation with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies, signals were detected using the SuperSignal Chemiluminescent Reagent (Pierce Chemical Co., Rockford, IL) with exposure of blots onto X-ray films.
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