Cells were lysed in 50 μl of cold 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 2 mM Na3VO4, 1× PhosSTOP EASYPACK, 1 mM Pefabloc, and 1× EDTA-free complete protease inhibitor cocktail (Roche, Basel, Switzerland) and then incubated for 10 min on ice. Then, cell debris was pelleted at 13,000 rpm at 4°C for 10 min. The soluble protein fraction was mixed with 4× Laemmli sample buffer (Bio-Rad, catalog no.1610747) and 2-mercaptoethanol (Sigma-Aldrich). Samples for Western blot were subjected to electrophoresis on 4 to 12% bis-tris gels (Invitrogen). To detect IRF5 dimers, we adopted Novex WedgeWell 14% tris-glycine gel (Invitrogen, catalog no. XP00140BOX) to electrophoresis of protein samples according to the manufacturer’s instruction. Proteins were transferred to polyvinylidene difluoride membrane, and immunodetection was performed as previously published (84 (link)). The antibodies used were from Cell Signaling Technology: IκBα (9242s), phospho-p38 (9215S), p38 (9212S), and phospho-p44/42 MAPK (ERK1/2) (9101S).
Wedgewell 14 tris glycine gel
Manufactured by Thermo Fisher Scientific
The WedgeWell 14% tris-glycine gel is a pre-cast polyacrylamide gel used for protein electrophoresis. The gel has a 14% acrylamide concentration and is designed for the separation of proteins in a tris-glycine buffer system.
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2 protocols using wedgewell 14 tris glycine gel
1
Protein Extraction and Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in 50 μl of cold 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 2 mM Na3VO4, 1× PhosSTOP EASYPACK, 1 mM Pefabloc, and 1× EDTA-free complete protease inhibitor cocktail (Roche, Basel, Switzerland) and then incubated for 10 min on ice. Then, cell debris was pelleted at 13,000 rpm at 4°C for 10 min. The soluble protein fraction was mixed with 4× Laemmli sample buffer (Bio-Rad, catalog no.1610747) and 2-mercaptoethanol (Sigma-Aldrich). Samples for Western blot were subjected to electrophoresis on 4 to 12% bis-tris gels (Invitrogen). To detect IRF5 dimers, we adopted Novex WedgeWell 14% tris-glycine gel (Invitrogen, catalog no. XP00140BOX) to electrophoresis of protein samples according to the manufacturer’s instruction. Proteins were transferred to polyvinylidene difluoride membrane, and immunodetection was performed as previously published (84 (link)). The antibodies used were from Cell Signaling Technology: IκBα (9242s), phospho-p38 (9215S), p38 (9212S), and phospho-p44/42 MAPK (ERK1/2) (9101S).
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