Purification of Nanobody Nb35 from E. coli

E. coli BL21 cells were transformed with the plasmid containing Nb35 target gene and were grown in TB medium with 100 μg/mL ampicillin, 1 M MgCl₂, 2% (w/v) glucose at 37 °C, 180 rpm for 3 h. IPTG (1 M) was added to induce the expression when OD600 reached 0.7–1.2. After 8 h expression, the cell pellet was collected and stored at −80 °C until use. Nb35 was purified by nickel affinity chromatography as previously described,^{25 (link)} followed by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 column (GE Healthcare). The column was pre-equilibrated with 20 mM HEPES, pH 7.4 and 100 mM NaCl. Glycerin 30% (v/v) was added to collect Nb35 which was frozen in liquid nitrogen and stored at −80 °C.