Mini protean tetra cell electrophoresis apparatus system

Western blot analysis was used to investigate the protein content for Pgm2p-GFP fusion protein. Different strains were grown in media treated with and without LiCl. Protein extraction was performed as described by Szymanski [26 ]. Bicinchoninic acid assay (BCA) was performed to estimate protein concentration as described by the manufacturer (Thermo Fisher^®). Equal amounts of total protein extract (50 μg) were loaded onto a 10% SDS-PAGE gel, run on Mini-PROTEAN Tetra cell electrophoresis apparatus system (Bio-Rad^®). Proteins were transferred to a nitrocellulose 0.45 μm membrane via a Trans-Blot Semi-Dry Transfer (Bio-Rad^®). Mouse monoclonal anti-GFP antibody (Santa Cruz^®) was used to detect protein levels of Pgm2p-GFP. Mouse anti-Pgk1 (Santa Cruz^®) was used to detect Pgk1 protein levels used as internal controls. Immunoblots were visualized with chemiluminescent substrates (Bio-Rad^®) on a Vilber Lourmat gel doc Fusion FX5-XT (Vilber^®). Densitometry analysis was carried out using the FUSION FX software (Vilber^®). Experiments were repeated at least three times; t-test analysis (P-value ≤ 0.05) was used to determine statistically significant results.

Western blot analysis was used to investigate the protein content for Pgm2p-GFP fusion protein. Deleted mutant strains with GFP-tagged PGM2 were grown in media treated with and without LiCl to investigate protein levels of PGM2. Protein extraction was performed as described by Szymanski et al. [75 (link)]. Bicinchoninic acid assay (BCA) was performed to estimate protein concentration as described by the manufacturer (Thermo Fisher^®, Ottawa, ON, Canada). Equal amounts of total protein extract (50 μg) were loaded onto a 10% SDS-PAGE gel, run on Mini-PROTEAN Tetra cell electrophoresis apparatus system (Bio-Rad^®). Proteins were transferred to a nitrocellulose 0.45 μm membrane via a Trans-Blot Semi-Dry Transfer (Bio-Rad^®). Mouse monoclonal anti-GFP antibody and anti-Pgk1 (Santa Cruz^®) were used to detect protein levels of Pgm2p-GFP and Pgk1p, respectively. Immunoblots were visualized with chemiluminescent substrates (Bio-Rad^®) on a Vilber Lourmat gel doc Fusion FX5-XT (Vilber^®). Densitometry analysis was carried out using the FUSION FX software (Vilber^®, Collégien, France). Experiments were repeated at least three times. T-test analysis (p-value ≤ 0.05) was used to determine statistically significant results.