Labeling of Recombinant Proteins

Labeling of recombinant probes was performed by conjugation of Meso Scale Discovery (MSD) Gold sulfo-TAG NHS-Ester and biotin to primary amines, as previously described [23 (link)]. Briefly, recombinant HIP protein was mixed with sulfo-TAG, incubated at RT for 2 h, and purified on a desalting spin column (Thermo Scientific, Waltham, MA, USA). Labeling efficiency was determined by spectrophotometry (450 nm), and protein concentration by a bicinchoninic acid (BCA) kit (Sigma Aldrich, St. Louis, MO, USA). Biotin labeling of PI HIP recombinant protein was performed using a biotinylation kit (Thermo Scientific), and products purified with a desalting spin column. Protein concentration and labeling efficiency were determined by spectrophotometry (500 nm) and a BCA kit, respectively.