After this step, it's crucial that SyrB1-Ppt-Thr remain at cool temperatures to prevent spontaneous hydrolysis of the thioester bond that links threonine to PPT-SyrB1. The amino acidloaded protein was then concentrated using the previous centrifugal filter unit, and buffer exchanged four times with RXN buffer to remove excess reaction components. The protein was aliquoted, flash frozen in liquid nitrogen, and stored at -80°C.