Cells were lysed in RIPA buffer supplemented with freshly added protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Twenty micrograms of protein evaluated using the BCA method (Sigma-Aldrich, St. Louis, MO, USA) was loaded onto a 12% SDS polyacrylamide gel (Bio-Rad, Hercules, CA, USA) followed by electrophoresis at a constant voltage of 100 V for 1.5 h. Proteins were transferred to a PVDF membrane (0.2 μm pore size, Merck Millipore, Burlington, MA, USA). The membranes were incubated for 40 min in blocking solution: 4% bovine serum (BSA; Sigma-Aldrich, St. Louis, MO, USA) in TBS-T buffer (25 mM Tris, 0.2 M glycine, 20% methanol, 1% Tween) and incubated overnight at 4 °C with primary and then with secondary antibodies (Supplementary Materials Table S2 ). The detection was performed using a Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) with the ChemiDoc detector (Bio-Rad, Hercules, CA, USA). Protein band intensities were quantified using ImageLab 5.2.1 software.
Chemidoc detector
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc detector is a compact and versatile imaging system designed for the detection and analysis of chemiluminescent and fluorescent signals in various life science applications. It utilizes a high-sensitivity camera and advanced optics to capture and document the results of protein and nucleic acid detection assays, such as Western blots, EMSA, and gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris gels 3 12 gradient gels, by Thermo Fisher Scientific (2 mentions)
Instantblue, by Abcam (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Bovine serum bsa, by Merck Group (1 mentions)
Bca method, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using chemidoc detector
1
Western Blot Analysis of Protein Lysates
2
MS2 VLPs Electrophoretic Analysis
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MS2 VLPs variants were analyzed by electrophoresis in both denaturing and native conditions. For SDS-PAGE, samples were separated on 12% gels Tris/Glycine gels using standard Laemmli protocol, whereas for non-denaturing electrophoresis Bis-Tris gels 3-12% gradient gels were used (Life Technologies), following the manufacturer’s recommendations. A Chemidoc detector (BioRad) was used for fluorescence detection with excitation at 546 nm. Gels were stained in InstantBlue (Expedeon).
3
MS2 VLPs Electrophoretic Analysis
4
Western Blot Analysis of Protein Expression
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Cells were lysed using RIPA buffer supplemented with Complete Protease Inhibitor Cocktail and PhosphoSTOP Phosphatase Inhibitor Cocktail. Cell extract concentration was measured using the Bicinchoninic Acid Protein Assay Kit following the manufacturer’s protocol. 20 µg of total protein extracts were loaded onto 12% Bis-Tris gels (TGX™ FastCast™ Acrylamide Kit) for protein separation and transferred to PVDF membranes (0.2 μm pore size). After blocking in 4% in TBST buffer for 40 min, the membranes were cut and incubated at 4 °C overnight with the following primary antibodies (Supplementary Data, Table S1 ). Secondary antibodies used for detection were HRP-conjugated (Supplementary Data, Table S1 ). The detection was performed using Clarity Western ECL substrate with the ChemiDoc detector (BioRad, Hercules, CA, USA). Band intensities were quantified using ImageLab 5.2.1 software. The original uncropped and unprocessed scans of all blots are in the raw data file.
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