N-cadherin Polyclonal Antibody

Western blotting N‐cadherin

Experiment
Western blotting N‐cadherin
Product
N-cadherin Polyclonal Antibody from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Protocol tips
-Rabbit (1:1000)

Publication protocol

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).

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Manufacturer protocol

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