Ez link sulfo nhs ss biotin solution

Mixed cortical-hippocampal neuronal cultures prepared from newborn WT or S2KO mice were plated on poly-D-lysine-coated plates and cell surface biotinylation was performed at DIV10-12. To do so, cells were cooled down on ice for 15 min and washed 3 times with PBS-Mg-Ca (PBS supplemented with 0.5 mM MgCl₂ and 1 mM CaCl₂). Next, cells were incubated on ice with EZ-Link Sulfo-NHS-SS-Biotin solution (Thermo Fisher Scientific; 0.5 mg/ml in PBS) for 25 min, washed 3 times with quenching buffer (40 mM glycine, 0.4% BSA in TBS-Ca-Mg) and 2 times with TBS-Ca-Mg. Cells were scraped in lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 2 mM EGTA, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, protease and phosphatase inhibitors) and the lysates were rotated for 1.5 hours at 4°C. After centrifugation (15 min, 14,000x g), the supernatants were used for pull down of biotinylated proteins with NeutrAvidin slurry (Thermo Fisher Scientific). Equal amounts of proteins were loaded on the slurry. After washing 3 times with lysis buffer, the beads were snap-frozen and stored at −80°C until mass spectrometry analysis or boiled in Laemmli buffer to release captured proteins for western blot analysis.