Cells were harvested and stored at −80°C. Crude lysates were prepared from NMuMG, MDA-MB-231, and MCF-10A cells as described previously [55 (link), 56 (link), 51 (link)]. Protein concentrations were determined, using the Bradford reagent (Bio-Rad), and 25–50 ug of proteins were resolved on NuPAGE Novex 4–12% Bis-Tris gels, transferred to PVDF membranes and probed with respective antibodies. Peroxidase conjugated respective secondary antibodies were used to label the proteins. The proteins were detected by enhanced chemiluminescence detection kit (SuperSignal West Pico; Pierce). Densitometric analysis was performed using imageJ analysis and results were corrected for protein loading by normalization for β-actin expression as described in [55 (link)].
Nupage novex 4 12 bis tris gels
Manufactured by Bio-Rad
Sourced in Germany
The NuPAGE Novex 4–12% Bis-Tris gels are pre-cast polyacrylamide gels designed for protein separation and analysis. They feature a Bis-Tris buffer system and a 4-12% gradient, allowing for the resolution of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Bradford reagent, by Bio-Rad (1 mentions)
Ecl western blot detection reagent, by GE Healthcare (1 mentions)
Anti mouse igg pod antibody, by Merck Group (1 mentions)
Anti β tubulin antibody, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab126061, by Abcam (1 mentions)
Nckap1, by Abcam (1 mentions)
Wave2, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
4 protocols using nupage novex 4 12 bis tris gels
1
Western Blot Analysis of Cell Lysates
2
Fibronectin and ED-B Protein Detection in Cell Lysates
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60 μg of protein from the respective cell lysates; 0.5, 2, or 5 μg of cellular fibronectin (Calbiochem, Bad Soden, Germany); and 50 ng each of 67B89 and 6789 were subjected to SDS-PAGE on NuPAGE Novex 4–12% BisTris gels and transferred to PVDF membranes (Bio-Rad) by electrophoresis. Membranes were blocked overnight at 4 °C using 5% dry milk in PBST, followed by incubation with 10 nm 77405 protein for 1 h at room temperature. ED-B binding was detected after incubation with a rabbit anti-Strep-tag IgG (Genscript, Piscataway, NJ) and an anti-rabbit IgG POD antibody (Sigma-Aldrich). Fibronectin was detected on separate membranes using a monoclonal anti-human fibronectin (R&D Systems, Wiesbaden-Nordenstadt, Germany) and an anti-mouse IgG POD antibody (Sigma-Aldrich). Following incubation with the substrate ECL Western blot detection reagent (GE Healthcare) for 1 min, blots were developed by 1 min of exposition. Antibodies were stripped from the membranes using 0.2 m glycine buffer, pH 2.2, supplemented with 0.1% SDS and 1% Tween 20. β-Tubulin was detected using an anti-β-tubulin antibody (Sigma-Aldrich) and an anti-mouse IgG POD antibody (Sigma-Aldrich).
3
Western Blot Protein Analysis
4
NCKAP1 Knockout in MEFs
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Lysates were collected on ice by scraping cells in RIPA buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1X protease and phosphatase inhibitors). The tubes were centrifuged for 10 min at 15,000 rpm and 4 °C. The lysate was transferred to a clean Eppendorf tube and measured using Precision Red.
The 40 μg of protein lysate was resolved on NuPAGE Novex 4–12% Bis-Tris gels and transferred onto a nitrocellulose membrane Bio-Rad system. Membranes were blocked with 5% BSA in TBS-T (10 mM Tris pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 20 min prior to overnight incubation with the primary antibody at 4 °C on a shaking incubator. Membranes were then washed three times for 5 min each in TBS-T. Membranes were then incubated at room temperature for 1 hour with secondary AlexaFluor conjugated antibodies, after which the blots were washed again for 5 min in TBS-T three times before being imaged on the Li-Cor Odyssey CLx machine. Images were then analysed using the Image Studio Lite Version 5.2. Lysates were harvested on 4 independent occasions from NCKAP1fl/fl and NCKAP1-/- MEFs.
Antibodies used: NCKAP1 (Abcam, Cambridge, UK: ab126061), CYFIP1 (Abcam: ab156016), WAVE2 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-33548) and α-Tubulin (Sigma, St. Louis, MO, USA; clone B512) as the loading control.
The 40 μg of protein lysate was resolved on NuPAGE Novex 4–12% Bis-Tris gels and transferred onto a nitrocellulose membrane Bio-Rad system. Membranes were blocked with 5% BSA in TBS-T (10 mM Tris pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 20 min prior to overnight incubation with the primary antibody at 4 °C on a shaking incubator. Membranes were then washed three times for 5 min each in TBS-T. Membranes were then incubated at room temperature for 1 hour with secondary AlexaFluor conjugated antibodies, after which the blots were washed again for 5 min in TBS-T three times before being imaged on the Li-Cor Odyssey CLx machine. Images were then analysed using the Image Studio Lite Version 5.2. Lysates were harvested on 4 independent occasions from NCKAP1fl/fl and NCKAP1-/- MEFs.
Antibodies used: NCKAP1 (Abcam, Cambridge, UK: ab126061), CYFIP1 (Abcam: ab156016), WAVE2 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-33548) and α-Tubulin (Sigma, St. Louis, MO, USA; clone B512) as the loading control.
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