Fluorescent Labeling of Human Transferrin

Human holo Tf (Sigma) was conjugated to Alexa Fluor 700 (AF700) or AF750 (Life Technologies) through monoreactive N-hydroxysuccinimide ester to lysine residues in the presence of 100 mM sodium bicarbonate, pH 8.3, according to manufacturer's instructions 14 (link). The probes were purified using Amicon Ultra-4 centrifugal filter units (MWCO 30 kDa). After extensive washes with phosphate buffered saline (PBS), the probes were reconstituted in PBS and protein concentration was normalized to 1 mg/mL 14 (link),22 (link). The degree of labeling of the probes was assessed by spectrophotometer DU 640 (Beckman Coulter, Fullerton, CA, USA). The average degree of labeling was no more than 2 fluorophores per Tf molecule. Custom Tf-QC-1 conjugation was performed by Li-Cor (Lincoln, NB, USA) with average dye to protein ratio of 3. All ligands were normalized to concentration 1 mg/mL in phosphate-buffered saline pH 7.6 and filter sterilized. In Figure S1, several fluorescently labeled or unlabeled Tf probes, kept at 4 °C for 2-6 weeks since conjugation, were subjected to immunoblotting analysis using anti-human Tf. All Tf probes displayed overall good protein stability when stored at 4 °C, for 2-6 weeks.