Excess streptavidin magnetic beads

l-AHA-labeled cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biosharp) containing protease inhibitor cocktail (TaKaRa). Proteins from cell lysis were collected and quantified using the bicinchoninic acid (BCA) protein quantification kit (Biosharp), and the protein concentration was adjusted. By clicking chemical reactions, proteins containing l-AHA were labeled using BPDO-PEG4000-biotin (MCE) with a final concentration of 40 μM. Excess streptavidin magnetic beads (YEASEN) were added to the protein mixture for adsorption of biotin-labeled proteins, and the unbound proteins were washed away with a RIPA buffer. The remaining beads and proteins were added with 40 μl 4× SDS-PAGE loading buffer (TaKaRa) containing 2 mM BPDO-PEG4000-biotin and incubated at 98°C for 10 min. Samples were stored at –80°C for subsequent detection.

l-AHA-labeled cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biosharp) containing protease inhibitor cocktail (TaKaRa). Proteins from cell lysis were collected and quantified using the bicinchoninic acid (BCA) protein quantification kit (Biosharp), and the protein concentration was adjusted. By clicking chemical reactions, proteins containing l-AHA were labeled using BPDO-PEG4000-biotin (MCE) with a final concentration of 40 μM. Excess streptavidin magnetic beads (YEASEN) were added to the protein mixture for adsorption of biotin-labeled proteins, and the unbound proteins were washed away with a RIPA buffer. The remaining beads and proteins were added with 40 μl 4× SDS-PAGE loading buffer (TaKaRa) containing 2 mM BPDO-PEG4000-biotin and incubated at 98°C for 10 min. Samples were stored at –80°C for subsequent detection.