Whole-cell protein lysates were prepared by lysing cells in Radioimmunoprecipitation assay buffer (RIPA) supplemented with cOmplete Protease inhibitor (Roche, Basel, Switzerland) and PhosphStop (Roche). Protein concentrations were determined by bicinchoninic acid assay (BCA, Thermo Fisher). 10 µg of protein were denatured in Laemmli buffer at 95 °C for 5 min. Lysates were loaded onto 16% or 10% Tris-Glycin (Thermo Fisher) for gel electrophoresis depending on the protein sizes of interest. Proteins were transferred onto Polyvinylidenfluorid (PVDF) membranes (Roche), blocked with 5% dry milk for 1 h, and incubated with primary antibodies overnight at 4 °C, then secondary antibodies for 1 h at room temperature. Chemiluminescent signal was detected using Enhanced chemiluminescence (ECL) Western Blotting Substrate (Thermo Fisher) and a Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) using ImageLab (v6.0.1). Quantification was performed with ImageJ (v.1.52a).
Tris glycin
Manufactured by Thermo Fisher Scientific
Tris-Glycin is a buffer solution used in various laboratory techniques, such as gel electrophoresis and Western blotting, to maintain a specific pH range during the separation and analysis of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Fusion fx7 imaging system, by Vilber (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Phosphstop, by Roche (2 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Enhanced chemiluminescence ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
2 protocols using tris glycin
1
Western Blot Protein Quantification
2
Whole-Cell Protein Extraction and Western Blot
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Whole-cell protein lysates were prepared by lysing cells in RIPA supplemented with cOmplete Protease inhibitor (Roche) and PhosphStop (Roche). Protein concentrations were determined by bicinchoninic acid assay (Thermo Fisher Scientific). 10 μg of protein were denatured in Laemmli buffer at 95°C for 5 minutes. Lysates were loaded onto 16%, or 10% Tris-Glycin (Thermo Fisher Scientific) gels for gel electrophoresis depending on the protein sizes of interest. Proteins were transferred onto polyvinylidene difluoride membranes (Roche), blocked with 5% dry milk or 5% BSA for 1 hour and incubated with primary antibodies overnight at 4°C, then secondary antibodies for 1 hour at room temperature. Chemiluminescent signal was detected using Enhanced chemiluminescence (ECL) Western Blotting Substrate (Thermo Fisher Scientific) and a Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France). Quantification was performed with ImageJ.
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