Mouse anti his tag

mAbs (21 μg) in sodium phosphate buffer (pH 7.0) were bound to Protein G beads (GE Healthcare). Unbound mAbs were removed by centrifuging and washing the beads with sodium phosphate buffer. HEK293T cells were transiently transfected to express the extracellular region of CSF-1R [23 (link)]. One milliliter of the HEK supernatant was incubated with the protein G + mAbs. The resin was washed 5 × with sodium phosphate buffer. The resin was incubated with 30 μl PBS + reducing sample buffer. This was incubated at 95°C for 5 min and was spun to precipitate the resin. The supernatant was loaded into a 10% SDS-PAGE gel. The western blot nitrocellulose membrane was probed with mouse anti-His tag (Invitrogen) and with goat anti-mouse, HRP-conjugated (Dako). N-linked oligosaccharides were removed from the extracellular region of CSF-1R using PNGase F (Promega). The purified extracellular region (50 μg in 12 μl of 0.5 M sodium phosphate buffer, pH 7.5) was mixed with 1 μl SDS (5%) and 1 μl DL-Dithiothreitol (DTT) (1 M). The sample was denatured at 95°C for 5 min and was cooled at room temperature for 5 min. Sodium phosphate buffer, NP-40 (10%) and PNGase F (2 μl each) were added to the mixture. This was incubated at 37°C for 2 h. Samples were resolved in a 10% SDS-PAGE gel and were analyzed by Ponceau S staining of the proteins transferred to a nitrocellulose membrane.