Western Blot Analysis Protocol
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Corresponding Organization :
Other organizations : University of Oklahoma Health Sciences Center
Protocol cited in 2 other protocols
Variable analysis
- Cells were washed once with pre-warmed 1X PBS and then lysed directly with ice-cold lysis buffer
- Whole cell lysates (50 μg) were resolved on a 10% SDS-PAGE and transferred to a nitrocellulose membrane
- The membrane was probed with various antibodies, including mouse polyclonal anti-FLAG antibody, mouse monoclonal anti-β-actin antibody, and mouse monoclonal anti-GAPDH antibody
- The nitrocellulose membrane was then probed with an anti-mouse HRP-conjugated secondary antibody
- The signal was detected using Supersignal West Dura with a BioRad ChemiDoc XRS imaging system
- Expression of protein and loading control densitometry analysis was performed using Image Lab v4.1 software
- Lysis buffer composition (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.5% sodium-deoxycholate, and Complete™ protease inhibitor cocktail)
- SDS-PAGE gel concentration (10%)
- Nitrocellulose membrane type (Santa Cruz Biotechnology, Inc., Dallas, TX)
- Primary antibody dilutions (mouse polyclonal anti-FLAG antibody (1:4000 dilution), mouse monoclonal anti-β-actin antibody (1:5000 dilution), and mouse monoclonal anti-GAPDH antibody (1:5000 dilution))
- Secondary antibody (anti-mouse HRP-conjugated)
- Detection method (Supersignal West Dura with a BioRad ChemiDoc XRS imaging system)
- Densitometry analysis software (Image Lab v4.1)
- Previous publications (32 and 33) that used similar immunoblotting protocols
- No negative controls were explicitly mentioned in the provided information
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