Lourmat gel doc fusion fx5 xt

Total proteins were purified in the absence of detergent [43 (link)]. Briefly, samples containing spores were pelleted by centrifugation and rinsed with 1× PBS. Total protein concentration was measured using the bicinchoninic acid assay (BCA) using protocols from the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were electrophoretically separated in 10% polyacrylamide-SDS gels using a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad^®, Hercules, CA, USA) [40 (link)]. The proteins were transferred to 0.45 μm nitrocellulose (Bio-Rad^®, Hercules, CA, USA) with a Trans-Blot Semi-Dry Transfer system (Bio-Rad^®, Hercules, CA, USA). Table 1 shows the tested antibodies. Following transfer, immunoblots were viewed with enhanced chemiluminescence (ECL) substrates (Bio-Rad^®, Hercules, CA, USA) using a Vilber Lourmat gel doc Fusion FX5-XT (Vilber^®, Marne-la-Vallee, France). Densitometry was conducted with FUSION FX software (Vilber^®, Marne-la-Vallee, France), using total protein to normalize measurements. Data represent three separate experiments from three different total protein isolations.

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.