For immunoblots 48-72 h after siRNA transfection (Supplementary Fig. 1), Cos-7 cells were lysed in radioimmunoprecipitation (RIPA) buffer (Sigma) supplemented with fresh proteases inhibitors (Sigma Aldrich 11836170001) on ice for 30 min. A centrifugation at 16,000g for 10 min at 4 °C was performed to remove the insoluble material. Protein concentrations were determined using a Pierce BCA protein assay kit (Life Technologies, 23227) and equal amounts of protein were analysed by self-casted 7.5% or 15% SDS-PAGE (30-50 μg of protein per lane). For immunoblotting, proteins were transferred to nitrocellulose membranes (BioRad) electrophoretically and incubated with the specified primary antibodies (see above), diluted in 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST). The blots were further incubated with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare) and visualized using ECL (GE Healthcare). Where required, images of western blotting were treated for contrast enhancement, and densitometric analyses were performed using ImageJ.
Fresh proteases inhibitors
Manufactured by Merck Group
Fresh proteases inhibitors are lab equipment designed to inhibit the activity of proteases, which are enzymes that break down proteins. They are used to maintain the integrity of protein samples during research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti mouse or anti rabbit horseradish peroxidase hrp conjugated secondary antibodies, by GE Healthcare (1 mentions)
Anti rabbit or anti mouse hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
2 protocols using fresh proteases inhibitors
1
Immunoblot Analysis Following siRNA Transfection
2
Immunoblot Analysis of siRNA-Transfected Cos-7 Cells
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For immunoblots 48-72 hours after siRNA transfection, Cos-7 cells were lysed in RIPA buffer (Sigma) supplemented with fresh proteases inhibitors (Sigma Aldrich 11836170001) on ice for 30 minutes.
A centrifugation at 16,000 g for 10 min at 4˚C was performed to remove the insoluble material. Protein concentrations were determined using a Pierce BCA protein assay kit (Life Technologies, 23227) and equal amounts of protein were analyzed by self-casted 7.5 or 15% SDS-PAGE (30-50 µg of proteins per lane).
For immunoblotting, proteins were transferred to nitrocellulose membranes (BioRad) electrophoretically and incubated with the specified primary antibodies (see above), diluted in 5% non-fat dry milk in Tris buffered saline with Tween 20 (TBST). The blots were further incubated with anti-rabbit or anti-mouse HRP conjugated secondary antibodies (GE Healthcare) and visualized using ECL (GE Healthcare). Where required, images of Western blotting were treated for contrast enhancement and densitometric analyses were performed using ImageJ.
A centrifugation at 16,000 g for 10 min at 4˚C was performed to remove the insoluble material. Protein concentrations were determined using a Pierce BCA protein assay kit (Life Technologies, 23227) and equal amounts of protein were analyzed by self-casted 7.5 or 15% SDS-PAGE (30-50 µg of proteins per lane).
For immunoblotting, proteins were transferred to nitrocellulose membranes (BioRad) electrophoretically and incubated with the specified primary antibodies (see above), diluted in 5% non-fat dry milk in Tris buffered saline with Tween 20 (TBST). The blots were further incubated with anti-rabbit or anti-mouse HRP conjugated secondary antibodies (GE Healthcare) and visualized using ECL (GE Healthcare). Where required, images of Western blotting were treated for contrast enhancement and densitometric analyses were performed using ImageJ.
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