Pierce bca protein reagent

Equine placental and testicular tissues were homogenized in buffer (0.1 M K 3 PO 4 , pH 7.4, 20% glycerol, 5 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride and 1 μg/μL aprotinin) and then briefly sonicated. Homogenates were centrifuged at 15,000 g for 10 min and supernatant was transferred to a new tube and centrifuged again at 100,000 g for 1 h. The resulting microsomal pellet was recovered and resuspended. The concentrations of crude protein were determined using the Pierce BCA Protein Reagent (Thermo Scientific). Microsomal preparations were stored in aliquots at -80°C.

Tissues were homogenized in 0.1 M KPO4 pH 7.4, 20% glycerol, 5 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride and 1 μg/μL aprotinin, and then briefly sonicated. Homogenate was centrifuged at 15,000 g for 10 min. Supernatant was transferred to a new tube and spun for 1 h at 100,000 g, and the resultant microsomal pellet was resuspended in buffer. Protein concentrations were determined using the Pierce BCA Protein Reagent (Thermo Scientific). Microsomal preparations were stored at -80°C.

Placental tissues were homogenized in buffer (0.1 M K 3 PO 4 pH 7.4, 20% glycerol, 5 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride and 1 μg/μL aprotinin) and then briefly sonicated. Homogenates were centrifuged at 15,000 g for 10 min to recover mitochondria and the supernatant was transferred to a new tube and centrifuged again at 100,000 g for 1 h to recover microsomes, as previously described and validated (Moran et al. 2002 , Corbin et al. 2016 (link), Legacki et al. 2017 , 2018 , Reynolds et al. 2018) . The resulting pellets (15,000 g -mitochondrial; 100,000 g -microsomal) were recovered and resuspended. The concentrations of crude protein in each were determined using the Pierce BCA Protein Reagent (Thermo Scientific). Microsomal preparations were stored in aliquots at -80°C.