Dynabeads streptavidin magnetic beads

The proteins samples used for immunoprecipitation (IP) were extracted from mouse BMDMs treated by tumor-supernatant (TS) alone or with bafilomycin (TS + Bafilo). Antibody immobilization was performed by incubating anti-LAMP2a (Hangzhou HUAAN Biotechnology, ET1601–24) with Dynabeads Streptavidin magnetic beads (Invitrogen, 65801D) in PBS at 4 °C for 4 h. After separating the antibody-coated beads by a magnetic rack (Bio-Rad) and 4–5 times washing, the coated beads were resuspended with protein extracts at 4 °C with continuous inversion for 8 h. Next, the IP products were separated and washed in a magnetic rack, with magnetic beads releasing by incubating in 0·1% SDS at 95 °C for 10 min and magnetic separation. The final products without beads were quantified by Bradford dye and analyzed by Western blot or mass spectrometry.
For mass spectrometry, the samples were subjected into NuPAGE Bis-Tris gels, followed by Coomassie Blue staining. Then gels were de-stained and cut into slices for subsequent reduction, alkylation and trypsin digestion. The extracted peptides were analyzed in Q Exactive Plus mass spectrometer (Thermo) and identified by database on Uniprot following standard procedures.

Cell lysate of HEK cells expressing TrkB-GFP, BirA and Bio-Kif-td was incubated with Dynabeads Streptavidin magnetic beads (Thermo Fisher). Dynabeads (25μl per 10cm plate of lysed HEK cells) were pre-incubated with blocking buffer (Tris 20 mM pH 8.0, KCl 150 mM, BSA 0.2%, Glycerol 0.2%). Lysates were then incubated with beads for 1 hour at 4°C and the beads were washed 3 times with wash buffer (Tris 25mM PH 8.0, NaCl 150mM, TX-100 0.1%). For western blot analysis, bound proteins were eluted by addition of 50μl SDS-PAGE sample buffer and heated to 95°C for 5 minutes.