The β-Lactoglobulin (Sigma-Aldrich, Oakville, Ontario, Canada) was dissolved in coupling buffer to a final concentration of 12 mg/ml β-Lactoglobulin-coupled gel matrix.
The samples were then drained and the beads were equilibrated in 3 mL coupling buffer before adding 1 mL 0.1 M sodium cyanoborohydride in coupling buffer and mixing overnight at 4 °C.
The columns were loaded with 500 μL of the bead slurry before equilibration with 6 mL coupling buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2).
The beads were then washed with coupling buffer and resuspended in blocking buffer (coupling buffer plus 0.2 M glycine).
A column prepared using coupling buffer that was lacking CS-A was used as a control.
CNBr-activated Sepharose was re-swollen in 1 mM HCl and rinsed with coupling buffer.
The following day the column was washed with coupling buffer to remove nonspecific antibodies.
After the coupling reaction, excess antigen was washed away with coupling buffer.
The beads were washed with a final wash of coupling buffer then spun down and resuspended in 1.0 M NaCl.
5 mg GST protein was dissolved in the coupling buffer (0.5 M NaCl, 0.1 M NaHCO3, pH 8.3).
The gel was then washed with sodium acetate buffer (0.1 M; pH 4.0), followed by coupling buffer.
