Coomassie blue r250

5 × 10⁵ of naïve and primed hESCs were counted and collected repeatedly for the following assays. Total RNA was isolated by EZ-press RNA Purification Kit (EZ Bioscience, #B0004DP), and its quality was determined using a Nano Drop 2000 (Thermo) and quantitated by absorbance at 260 nm. For protein extraction, hESCs were lysed with SDS Lysis Buffer (Beyotime, #P0013G) and centrifuged at 12,000 g for 5 min. Then, the supernatant was collected and the protein concentration was determined with BCA Protein Assay Kit (Bio-Rad, #P0011) according to the manufacturer's instructions. In parallel, total proteins were subjected to SDS-PAGE and visualized by Coomassie Blue staining (0.1% Coomassie blue- R250 (Sangon Biotech, #A100472), 10% acetic acid, 25% isopropanol) after cells were treated with CHX for 12 h. Then, the overall band intensities representing total protein levels in individual lanes were quantified by the ImageJ software.

The protein samples were analyzed by SDS polyacrylamide gel electrophoresis [25 (link)]. SDS-PAGE gels were prepared using a 5% stacking gel and a 15% separation gel (MDBio, Taiwan, China). The proteins were visualized by staining with Coomassie Blue R-250 (Sangon Biotech, Qingdao, China).
Western blot analysis was performed using Rb c-phycocyanin (Maded by Boster Biological Technology co.ltd, Qingdao, China) and peroxidase-conjugated goat anti-rabbit IgG (Sangon Biotech, Qingdao, China) as primary and secondary antibodies, and the dilution factors were 1:1000 (primary antibodies) and 1:2000 (secondary antibodies). The expression of phycocyanin was judged by observing whether there is a hybridization strip in the nitrocellulose membrane.