Following siRNA treatment, cells were pelleted and lysed in RIPA buffer. Protein concentration was determined by BCA assay (Thermo Fisher). Following SDS-PAGE, protein was transferred to Amersham PVDF membrane (Genesee Scientific) using a BioRad Trans-Blot SD semi-dry transfer unit. Blots were then blocked in milk for one hour at room temperature or overnight at 4°C. Incubation with primary antibody took place for one hour at room temperature or overnight at 4°C. Antibodies were diluted in 5% milk, with 0.1–0.2% Tween-20. Antibodies used were TWIST 2c1a (Santa Cruz Biotechnology, Dallas, TX) at 1:250–1:500 dilution, β-Actin, A1978 (Sigma Aldrich, St. Louis, MO) at 1:2500–1:5000 dilution; and Horseradish Peroxidase (HRP) conjugated anti-mouse secondary antibodies. For film-based westerns, Blue Devil film (Genesee Scientific) and ECL Plus chemiluminescent substrate (Thermo Fisher) were used to detect protein. For digital westerns, the Syngene Pxi4 digital blot imager and Michigan Diagnostics FemtoGlow chemilumescent substrate were used.
Blue devil film
Manufactured by Genesee Scientific
Blue Devil film is a precision laboratory film used for scientific and research applications. It provides high-quality recording and documentation of experimental data and observations. The core function of Blue Devil film is to serve as a reliable medium for capturing and preserving visual information in a laboratory setting.
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Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (2 mentions)
Ecl plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
β actin a1978, by Merck Group (1 mentions)
Trans blot sd, by Bio-Rad (1 mentions)
Twist 2c1a, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Anti β actin a1978, by Merck Group (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
2 protocols using blue devil film
1
Western Blot Analysis of TWIST Expression
2
Western Blot Analysis of TWIST Protein
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Cells were seeded at 250,000 cells per well in 6-well tissue culture plates and treated as described in Section 2.1 . Cells were pelleted and lysed in RIPA buffer, and protein concentration was determined using a BCA Assay (Thermo Fisher Scientific, Waltham, MA). 30 μg total protein per lane was run on 4% stacking and 10–12% resolving polyacrylamide gels and transferred to Immobilon-P PVDF membrane (Millipore, Billerica, MA) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA). Membranes were blocked with 5% dry milk dissolved in 1X PBS with 0.1% Tween-20. Antibodies were diluted in blocking buffer. Antibodies used were anti-TWIST, TWIST 2c1a (Santa Cruz Biotech, Dallas, TX); anti-β-Actin, A1978 (Sigma Aldrich, St. Louis, MO); and Horse Radish Peroxidase (HRP) conjugated anti-mouse secondary antibodies. ECL Plus chemiluminescent substrate (Pierce, Thermo Fisher Scientific, Waltham, MA) and Blue Devil Film (Genesee Scientific, San Diego, CA) were used for capturing images.
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