GFP-specific nanobody39 (
link) expression was performed in
E. coli (JM109). Expression was induced with 0.5 mM of isopropyl beta-D-1-thiogalactopyranoside (IPTG, ROTH), and cells were incubated at 37 °C for 24 h. Cells were lysed in the presence of
Lysozyme (100 μg ml
−1; Serva, Germany),
DNase (25 μg ml
−1; Applichem, Germany) and phenylmethanesulfonyl fluoride (2 mM,
PMSF, Sigma) followed by sonication (Branson
® Sonifier; 16 × 8 sec, 20% Amplitude) and debris centrifugation at 20000 x g for 30 min. Protein purification was performed with an
Äkta FPLC system (GE Healthcare, USA) using a 5 mL
His-Trap column (GE Healthcare, USA); peak fractions were concentrated to 2 ml using
Amicon filter columns (cut-off 10 kDa, Merck Millipore, Germany) followed by size exclusion chromatography using a
Superdex 75 column (GE Healthcare, USA). Peak fractions were pooled and protein aliquots were shock-frozen and stored at −80 °C. 1 mg purified nanobody protein was then labeled with
ATTO 647N (ATTO-TEC, Germany) with a theoretical DOL (degree of labeling) of 3, according to the manufacturers’ instructions. Unbound dye was removed by gel filtration in
PD10 columns (GE Healthcare, USA). The final
ATTO 647N labelled GFP-specific chromobody was obtained and preserved in PBS in the concentration of 1 mg ml
−1.
Chiu H.Y., Deng W., Engelke H., Helma J., Leonhardt H, & Bein T. (2016). Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time. Scientific Reports, 6, 25019.
