4.9. Western Blot Analysis

The preoptic areas of the hypothalamus from six lactating and six pup-deprived rat mothers were obtained for western blotting. Purification of proteins was carried out by radioimmunoprecipitation assay (RIPA) buffer. Then, the samples were centrifuged at 12,000× g for 30 min at 4 °C. Protein measurement was performed with BCA kit (Sigma-Aldrich, cat. no. BCA1-1KT). Proteins (25 μg per lane) were separated by standard SDS-PAGE and transferred to nitrocellulose (Bio-Rad, Cat. No. 1620112). Anti-Androgen receptor antibody (Sigma Aldrich, Cat. No. 06–680) was applied at a ratio 1:1000 and incubated at 4 °C for a day. The bound antibodies were labeled with anti-rabbit (1:2000; Jackson ImmunoResearch, Cat. No. 711035152) horseradish peroxidase-conjugated secondary antibody followed by visualization with Gel Doc XR+ imaging system (BioRad) using Clarity Western ECL Substrate (BioRad Laboratories, Cat. No. 170–5060). The molecular weight of the labeled proteins were measures with PageRuler Prestained Protein Ladder (cat. no. 26616, Thermo Scientific, Waltham, MA, USA).