Mass spectrometry analysis of FLAG-5MP complexes

FLAG-tagged 5MP (h5MP1/BZW2 from human and 5MP/Kra from fly) was ectopically expressed in relevant host cells (HEK293T for human and S2 for fly) by transient transfection, and one-step affinity-purified by anti-FLAG affinity resin.The one-step affinity purification products were resolved by SDS-PAGE, silver-stained and subjected for MS after dividing the whole lane into 12-15 fractions.