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.
Twist 2c1a
Manufactured by Santa Cruz Biotechnology
The TWIST 2c1a is a laboratory instrument designed for DNA and RNA manipulation. It provides precise and efficient processing of samples for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (2 mentions)
Trans blot sd, by Bio-Rad (2 mentions)
Ecl plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
β actin a1978, by Merck Group (1 mentions)
Blue devil film, by Genesee Scientific (1 mentions)
Ecl plus, by Thermo Fisher Scientific (1 mentions)
Gfp b 2, by Santa Cruz Biotechnology (1 mentions)
Nf κb p65 f 6, by Santa Cruz Biotechnology (1 mentions)
Gapdh 6c5, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
4 protocols using twist 2c1a
1
Western Blot Analysis of TWIST Expression
2
Western Blot Analysis of Protein
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Protein was run on 10% resolving polyacrylamide gels and transferred to PVDF membrane. Membranes were rinsed with PBS and blocked in 5–10% milk, 1 h at room temperature or overnight at 4 °C. Membranes were then incubated with mouse primary antibody in milk with Tween-20 (Ab Buffer) for 1 h at room temperature or overnight at 4 °C, and washed in PBS with 0.1% Tween-20 (PBST). Membranes were then incubated with anti-mouse secondary antibody in Ab Buffer for 1 h at room temperature, followed by an additional five PBST washes. Primary antibodies were: TWIST1, TWIST 2c1a (Santa Cruz Biotechnology sc-81417) 1:250-1:500; for RELA, NF-κB p65 F-6 (Santa Cruz Biotechnology sc-8008) 1:250-1:500; for GFP, GFP B-2 (Santa Cruz Biotechnology sc-9996) 1:1000; for actin, Sigma Aldrich A1978 or 2066. Secondary antibodies were HRP conjugated anti-mouse and anti-rabbit. Protein was detected using Blue Devil Film (Genesee) and ECL Plus (Thermo Fisher) or digital imaging. Quantitation of digital images was performed using the accompanying software from Syngene (Frederick, MD) or Carestream MI (Woodbridge, CT).
3
Protein Expression Analysis Protocols
4
Western Blot Analysis of TWIST and β-Actin
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