Chemiluminescent western blotting substrate

Yeast strains expressing chromosomally C-terminally tagged GFP fusion proteins Fas1, Fas2, Elo1, Elo2, Elo3, and Ole1-GFP were grown overnight in YPD, washed, inoculated at A₆₀₀ = 0.25 in −I/−C in the presence or absence of 2 mm Hcy, and cultivated for an additional 6 h. 3 A₆₀₀ units of logarithmically growing cells were harvested, and whole-yeast cell extracts were prepared by the method of Horvath and Riezman (56 (link)). Proteins were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes (Bio-Rad). Mouse monoclonal anti-GFP antibody (Roche Applied Science) followed by horseradish peroxidase–conjugated anti-mouse IgG (Pierce) secondary antibody was used to detect GFP fusion proteins. Rabbit polyclonal anti-GAPDH antibody (57 (link)) followed by horseradish peroxidase–conjugated anti-rabbit IgG (Pierce) secondary antibody was used to detect GAPDH, which served as loading control. Chemiluminescent Western blotting substrate (Thermo Scientific) was used for detection.

Purified apo secretagogin was analyzed using SDS-PAGE electrophoresis and Western blotting. Apo- secretagogin (1 mg/mL) was mixed with Laemmli sample buffer (0.5 M Tris-HCl pH 6.8, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue) and 5% (v/v) DTT at a range of concentrations (20 mM, 10 mM, 7 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, 0 mM). Samples were incubated overnight at room temperature and 2 μL (2 μg) was loaded onto a 12% polyacrylamide gel. The gel was run for 1 h at 120 V in 1× running buffer (25 mM Tris-HCl pH 8.3, 192 mM glycine, 0.1% (w/v) SDS), transferred onto PVDF membrane for 90 min at 320 mA in 1× transfer buffer (48 mM Tris, 39 mM Glycine, 0.037% (w/v) SDS, 20% (v/v) methanol), and incubated overnight at 4°C with rabbit polyclonal anti-secretagogin (Sigma-Aldrich, HPA006641) diluted 1:20,000 in blocking solution (5% (w/v) skimmed milk, 10 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween-20). Protein bands were visualised using chemiluminescent Western blotting substrate (Thermo Scientific) and exposing the membrane to X-ray film in the darkroom. Precision Plus Protein^™ Dual Color Standard from BioRad was used.

Yeast wildtype and sah1 mutant cells were transformed by the method of Gietz et al. (50 (link)). Transformants were selected for uracil prototrophy on SD−Ura medium. To detect expression of the GST fusion proteins, transformants were inoculated from overnight SD−Ura cultures in 5 ml of SD−Ura containing or not 0.05 mm CuSO₄ at A₆₀₀ = 1. Cells were collected after cultures reached A₆₀₀ = 10, and whole-yeast cell extracts were prepared by the method of Baerends et al. (51 (link)). Proteins were separated on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad). Goat anti-GST antibody (Sigma) followed by horseradish peroxidase-conjugated anti-goat IgG (Pierce) secondary antibody was used to detect GST fusion proteins. Chemiluminescent Western blotting substrate (Thermo Scientific) was used for detection.

BRIN-BD11 cells were analyzed using SDS-PAGE electrophoresis and Western blotting. Total protein (30 μg) was mixed with Laemmli sample buffer (0.5 M Tris-HCl pH 6.8, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue) and 5% (v/v) DTT at a range of concentrations (20 mM, 10 mM, 5 mM, 1 mM, 0 mM). Samples were incubated overnight at room temperature and loaded onto a 12% polyacrylamide gel. The gel was run for 1 h at 120 V in 1× running buffer (25 mM Tris-HCl pH 8.3, 192 mM glycine, 0.1% (w/v) SDS), transferred onto PVDF membrane for 90 min at 320 mA in 1× transfer buffer (48 mM Tris, 39 mM glycine, 0.037% (w/v) SDS, 20% (v/v) methanol), and incubated overnight at 4°C with rabbit polyclonal anti-secretagogin (Sigma-Aldrich, HPA006641) diluted 1:10,000 in blocking solution (5% (w/v) skimmed milk, 10 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween-20). Protein bands were visualised using chemiluminescent Western blotting substrate (Thermo Scientific and Roche, as indicated) and exposing the membrane to X-ray film in the darkroom. Precision Plus Protein^™ Dual Color Standard from BioRad was used.