Protein lysates were obtained from fibroblasts seeded on soft plates at passage 3. Protein lysates were processed according to standard procedures. In brief, cells were washed with PBS, lysed in radioimmunoprecipitation assay buffer (1% Triton X-100, 50 mM Tris, pH 7.5, 1 mM EDTA, and 150 mM NaCl) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich), and boiled in Laemmli buffer for 5 min. The samples were separated in SDS-PAGE gradient gels (4–15%), transferred to a nitrocellulose membrane using the BioRad system, and blocked in 5% nonfat dried milk dissolved in PBS supplemented with 0.1% Tween for 30 min at RT. The membranes were incubated with primary antibodies overnight at 4°C followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at RT. Immunoreactive bands were detected using an ECL-plus kit (Roche). Quantifications were done using ImageJ (National Institutes of Health) by normalizing the protein amount to α-tubulin or GAPDH amounts (loading controls). Antibody description and working dilutions can be found in Table S1.
Ecl plus kit
Manufactured by Roche
The ECL-plus kit is a chemiluminescent detection system used for the sensitive and quantitative analysis of proteins in Western blot applications. The kit provides the necessary reagents to perform enhanced chemiluminescence (ECL) detection of proteins labeled with enzyme-conjugated antibodies.
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Lab products found in correlation
2 protocols using ecl plus kit
1
Fibroblast Protein Extraction and Analysis
2
Western Blot Detection and Visualization
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Cells were washed with PBS on ice and immediately lysed in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue). The samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 10% gels, transferred to nitrocellulose membrane, and blocked with blocking solution (5% non-fat dried milk in PBS supplemented with 0.1% Tween detergent) for 30 min. The membranes were incubated with primary antibodies for 1 h at RT or overnight at 4 °C, followed by incubation with Horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Immunoreactive bands were detected using an ECL-plus kit (Roche), and chemiluminescence detection was performed using either film or a BioRad ChemiDoc MP instrument.
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