Accunanobeads

Prior to labeling, U2OS cells were treated with the indicated reagents and biotin phenol (Iris-Biotech, 41994-02-9) containing medium was further added to 250 µM final concentration and treated for 30 min. Then 1 mM hydrogen peroxide was added to the medium to activate APEX labeling reaction for 1 min, followed by immediate quenching of reaction with ice-cold quenching buffer (1xPBS, 10 mM sodium azide, 10 mM sodium ascorbate, 2.5 mM Trolox). After four washes with cold quenching buffer, the cells were collected from plates with scrapers. Cells were lysed in lysis buffer (100 mM NaPO4, PH 8.0, 8 M Urea, 1% SDS, 10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox, 10 mM TCEP) and passed through an insulin syringe for 15 times to break DNA. After sonication at water bath sonicator for 10 mins, protein lysates are cleared by centrifuge. Protein concentration was measured using 2-D quant kit (GE healthcare, Cat# 80648356), by following manufacturer' instruction. After alkylation with 20 mM iodoacetamide for 15 min, 1 mg of protein samples were aliquoted and equilibrated to the same volume with lysis buffer. After dilution with equal volume of ddH2O to reduce the concentration of urea to 4 M and SDS to 0.5%, the samples were incubated with streptavidin magnetic AccuNanobeads (Bioneer, Cat# TA-1015-1) at 4 o C overnight.