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Ecl prime chemiluminescence substrate

Manufactured by Merck Group

ECL Prime chemiluminescence substrate is a laboratory reagent used to detect and quantify proteins in Western blot analyses. It produces a luminescent signal in the presence of the target protein, which can be measured using a luminometer or imager.

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2 protocols using ecl prime chemiluminescence substrate

1

Analyzing CAP and PSP94 Protein Expression and Secretion

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To analyze the expression and secretion of CAP proteins, they were tagged with a hemagglutinin (HA) tag (YPYDVPDYA) and PSP94 was tagged with a FLAG tag (DYKDDDDK). Epitope-tagged proteins were extracted from 3 A600nm units of cells with 0.185 M NaOH (62 (link)), followed by precipitation with 10% TCA. In order to analyze their secretion into the culture medium, total proteins from 20 ml culture medium were precipitated with 10% TCA, acetone washed, solubilized in sample buffer, and analyzed by SDS-PAGE and Western blotting. Western blotting was performed using rat anti-HA antibody (rat, 1:2000, Roche #11867423001) and FLAG monoclonal antibody (mouse, 1:5000, Invitrogen #13-2500). As secondary antibodies: Goat Anti-Rat IgG antibody, Horseradish Peroxidase (HRP) conjugate (1:10000, Merck #AP136P), and Goat Anti-Mouse IgG (HRP) conjugates (1:10000, Bio-Rad #1706516) were employed. ECL Prime chemiluminescence substrate (Sigma-Aldrich) was used for signal development, and chemiluminescence was detected with an ImageQuant LAS 4000 biomolecular imager (GE Healthcare). Experiments were performed in triplicates with similar results.
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2

Time-course Analysis of Protein Expression

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Cells were grown overnight in SC raffinose media, diluted to 1 OD600 ml in SC galactose media, and cells corresponding to 3 OD600 units were harvested at 0-, 2-, 4-, 6-, and 21-h time points. Samples were extracted by NaOH, and proteins were precipitated using TCA and resuspended into sample buffer (Horvath and Riezman, 1994 (link)). Proteins were separated using SDS-PAGE and detected using a monoclonal antibody against GFP (mouse, 1:2,000 dilution, no. 11814460001; Roche Diagnostics) or Kar2 (rabbit, 1:5,000 dilution; Randy Schekman, University of California, Berkeley, Berkeley, CA) and using goat anti–rabbit IgG (H+L)-HRP conjugate (1:10,000 dilution, no. 1706515; Bio-Rad) and goat anti–mouse IgG (H+L)-HRP conjugates (1:10,000 dilution, no. 1706516; Bio-Rad). Subsequent detection occurred via peroxidase-coupled secondary antibodies and ECL Prime chemiluminescence substrate (Sigma-Aldrich) using an ImageQuant LAS 1000 biomolecular imager (GE Healthcare) and ImageQuant software. Western blot experiments were performed at least twice with similar results.
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