Sephacryl s 300

The egg yolks from hyperimmunized hens were physically separated from the egg white and first mixed gently with eight volumes of cold distilled water (acidified with 0.1 M HCl to give pH 4.0) to avoid possible disruptions of egg yolk granules due to the presence of high concentrations of acid. Cold acidified distilled water (pH 2.0) was then added to make a final dilution of 1:10. After mixing well, the mixture was adjusted to a pH 5.0-5.2 and incubated at 4 °C for 12 h. The WSF was obtained by centrifugation at 3.125 x g at 4 °C for 20 min. The supernatant was collected as the IgY rich WSF and titrated by indirect ELISA (mentioned below) using Sigma gliadin as a coating antigen. The WSF (10 mg protein/ml) of high titre was further purified by using a 1.0 x 110 cm column of Sephacryl S-300 (GE Healthcare, Piscataway, NJ) which was equilibrated and eluted with PBS at a flow rate of 3 ml/h. Blue dextran (Pharmacia Biotech Inc., Baie-d’Urfe, QC) and titrated water were used to determine void volume (V_o) and total volume (V_t) of the column, respectively. The partition coefficient was calculated from the formula: K_av = (V_e - V_o)/(V_t - V_o), in which V_e represents the volume of the peak fraction. The eluates (1 ml) were analyzed for IgY activity at 405 nm by ELISA. The eluates of IgY were pooled, freeze-dried and analyzed for protein content, total IgY and specific IgY.

Full length LCN-2 cDNA was synthesized by GeneScipt, USA. It was subcloned in pET28a vector at NdeI and XhoI restriction site. The construct was transformed into E.coli DH5-α cells for amplification and E.coli Rosetta for expression. A single colony was grown overnight as a mother culture. 10% of mother culture was inoculated and grown to 0.8–1.0 OD and induced with 0.5 mM IPTG for 2 h at 37 °C. The cells were then pelleted by centrifugation at 6000 rpm for 10 min at 4 °C in a microfuge, resuspended in 10% volume of 20 mM Tris pH 8.0, containing 300 mM NaCl and 10% Glycerol. The mixture was sonicated for 30 s on and off each for 6 cycles, and then centrifuged at 12000 rpm for 30 min at 4 °C. The supernatant fraction was passed over a Nickel NTA (BioVision, USA) column as per the manufacturer’s protocol. The column was washed twice with 10 times the bed volume with 20 mM Tris pH 8.0, with 300 mM NaCl, 10% Glycerol and 20 mM Imidazole. The protein was eluted with 20 mM Tris pH 8.0, 300 mM NaCl, 10% Glycerol and 300 mM Imidazole with approximately five times the bed volume in multiple fractions. The protein was polished over Sephacryl S-300 (GE Healthcare, USA, GE17–0599–10) following overnight dialysis at 4 °C in 1X PBS and 50% Glycerol. The filter (0.25 micron) sterilized protein was stored at −20 °C in working aliquots.