Bicinchoninic acid kit for protein determination bca1 1kt

Immunoblotting of semen samples was carried out as previously described [70 (link)]. The protein concentration was determined by a detergent compatible protein assay (Bicinchoninic Acid Kit for Protein Determination BCA1-1KT, Sigma). Samples (50 µg) were loaded on 10% Tris–HCl gels, electrophoretically separated, and electroblotted onto PVDF membrane (Bio-Rad Laboratories, CA, USA). The membranes were blocked with 5% non-fat dry milk in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h to reduce non-specific binding. The membranes were incubated at + 4 °C overnight with primary antibodies (Table 1), washed in TBS-T for 1 h then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. Following the final wash in TBS-T, the membranes were incubated with the Super Signal Chemiluminescence (CL)-HRP substrate system (Thermo Scientific, USA) for 5 min and the signals on the membranes were transferred to the hyperfilm (Amersham Biosciences; Buckinghamshire, England) in the dark room. Protein levels of syncytin 1, syncytin 2, SLC1A5, and MFSD2 were compared semiquantitatively with ImageJ 1.46 (NIH) analysis by normalizing to GAPDH.