Cell lysates were extracted using radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). Protein concentrations were determined using the bicinchoninic acid method (Sigma-Aldrich; EMD Millipore). Cell lysates containing 40 µg protein were loaded and separated on 10% SDS-PAGE gels and subsequently transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked in Tris Buffered Saline with 5% (w/v) skimmed milk and 0.05% Tween 20 (Thermo Fisher Scientific, Inc.) for 1 h at 37°C. Primary antibodies were incubated overnight at 4°C. The primary antibodies and mouse monoclonal anti-β-actin were purchased from Abcam (anti-Snail antibody; ab180714; diluted at 1:1,000; anti-Slug antibody; ab27568; diluted at 1:1,000; anti-N-cadherin antibody; PA5-19486; diluted at 1:1,000; anti-β-actin antibody; ab8226; diluted at 1:2,000) The pH2AX antibody (MBS837487; diluted at 1:2,000) was purchased from MyBioSource, Inc. (San Diego, CA, USA). The membranes were washed and incubated with secondary antibody (ab6721; Abcam) at 1:5,000 dilution for 2 h at room temperature. The membranes were washed again and developed using enhanced chemiluminescence substrate (Sigma-Aldrich; EMD Millipore). Quantitative analysis was performed using QuantiOne imaging software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Full paper
or join for free
to view the full paper.
Check out videos that might be relevant for performing Western blotting N‐cadherin using N-cadherin Polyclonal Antibody from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.