After overnight incubation at 4°C, the solution was subjected to a column packed with 5 ml monomeric avidin UltraLink™ resin (Pierce Biotechnology Inc., Rockford, IL, USA). Unlabeled protein and birA were removed by washing for 10 column volumes with buffer A before biotin labeled TLX LBD was eluted using buffer A supplemented with 2 mM biotin. The product was then concentrated and subjected to size exclusion chromatography using a 10/30 Superdex75™ column equilibrated and run in HTRF assay buffer.