Samples were resolved by standard reducing SDS–PAGE analysis on a 4–12% Bis-Tris gel (Thermo Fisher Scientific) in MES buffer and transferred to a PVDF membrane (Osmonics, GE Healthcare) by wet transfer at 100 V for 1 h in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol). Membranes were blocked with a 5% (v/v) skim milk powder in 0.1% (v/v) Tween-20/PBS for 1 h at room temperature. Primary antibody was added to the membrane in 5 ml blocking buffer and incubated overnight at 4°C in a capped tube, on rollers. Membranes were washed for 30 min at room temperature with 0.1% (v/v) Tween-20/PBS, followed by incubation with HRP-conjugated secondary antibody for 1 h at room temperature, which was diluted in 5 ml blocking buffer. The 30-min washing step was repeated once more, and antibody binding was visualized using the Luminata ECL system (Millipore) following the manufacturer's instructions on a ChemiDoc Touch Imaging System (Bio-Rad).
Luminata ecl system
Manufactured by Merck Group
The Luminata ECL system is a laboratory equipment used for Western blot analysis. It functions to detect and quantify specific proteins in biological samples through chemiluminescent detection.
Automatically generated - may contain errors
2 protocols using luminata ecl system
1
Western Blot Protein Detection Protocol
2
Western Blot Analysis of Smchd1 Protein
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Samples were resolved by standard reducing SDS-PAGE analysis on 4–12% Bis-Tris gels (Thermo Fisher Scientific) in MES buffer and transferred to a PVDF membrane (Osmonics, GE Healthcare) by wet transfer at 100 V for 1 h in transfer buffer [25 mM Tris, 192 mM glycine, 20% (v/v) methanol]. Membranes were blocked with a 5% (v/v) skim milk powder in 0.1% (v/v) Tween-20/PBS for 1 h at room temperature. Primary antibody (monoclonal in-house anti-Smchd1, clone 1D6, 1:2000 or anti-Tubulin SantaCruz Biotechnology, SC-23948, 1:5000) was added to the membranes in 5 mL blocking buffer and incubated overnight at 4 °C in a capped tube, on rollers. Membranes were washed for 30 min at room temperature with 0.1% (v/v) Tween-20/PBS, followed by incubation with secondary antibody (anti-rat IgG HRP-conjugated, Southern Biotech, 3030-05, 1:10,000, goat anti-mouse IgG HRP-conjugated, Southern Biotech, 1036-05, 1:10,000) for 1 h at room temperature, which was diluted in 5 mL blocking buffer. The 30 minute washing step was repeated and antibody binding was visualized using the Luminata ECL system (Millipore) following the manufacturer’s instructions.
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