Superose 6 10 300 size exclusion column

For size estimations, 500 μL of the eluted protein was loaded onto Superdex 200 10/300 size exclusion column (GE Healthcare) or Superose 6 10/300 size exclusion column (GE Healthcare). Fractions of 500 μL were collected from 7 or 8 mL to 24 mL. Fractions were analyzed by SDS-PAGE to identify factions containing the protein of interest and with EMSAs to determine DNA-binding activity. Size exclusion standards (Bio-Rad) ranging from 1.35 to 670 kDa were used to calculate the partition coefficients and estimate the sizes of the protein complexes.

To compare the k_off of FN-AFFUD and FN-AFBbk32 by a method that does not require addition of unlabeled ligand, 400 nM FN and 40 nM AFFUD or AFBbk32 were mixed together in TBS containing 100 mM NaCl and separated on a Superose 6, 10/300 size exclusion column (GE Healthcare Life Science) at a rate of 0.5 ml/min. Fractions of 0.5 ml were collected and assayed. FN was monitored by absorbance at 280 nm by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE) and AFpolypeptide by Tecan Genios Pro microplate reader with excitation 485 nm and emission 535 nm. FN, AFFUD or AFBbk32 was analyzed by itself as a control. The k_off of FN-AFFUD complex was estimated by estimating the lower limit of half-life (t_1/2) of complex decay and the following equation:
$t_{1/2} = \frac{0.693}{k_{off}}$