Purification of Sunflower Allergen Hel a 6

All the chromatographic procedures were performed using an ÄKTAprime plus (GE Healthcare Life Sciences) FPLC system^{14 (link)}. The crude pollen extract (5 mg/ml) was fractionated in HiTrap Q HP column (GE Healthcare Life Sciences) equilibrated with the same buffer followed by elution of column-bound proteins using a linear gradient of 0.1–1 M NaCl. Eluted fractions containing the desired protein at a higher level of purity were pooled, dialyzed against 20 mM phosphate buffer (pH 7.4), and then concentrated in < 10 kDa cut-off Amicon Ultrafiltration device (Millipore). About 2 mg of concentrated protein was loaded onto a Superdex 75 10/300 GL gel filtration column (GE Healthcare Life Sciences)^{15 (link)}. Eluted fractions were screened by IgE-western blot using serum of 4 sunflower-sensitized patients to check for the presence of Hel a 6. The fractions containing Hel a 6 at > 90% purity were pooled, dialyzed against 1 M ammonium bicarbonate buffer (pH 8.5), and lyophilized.