Rabbits were initially immunized with 200 μg full-length His::TEV tagged PALS-5 antigen in Freund's Complete Adjuvant. Rabbits were then subsequently boosted with four separate immunizations of 100 μg antigen in Freund's Incomplete Adjuvant over a 16-week period. Approximately 25 ml of serum was collected and PALS-5 polyclonal antibody was purified with an immuno-affinity chromatography column by cross- linking PALS-5 to cyanogen bromide (CNBr)-activated Sepharose 4B gel. Antibody was eluted from the affinity column in 100 mM glycine buffer pH 2.5, precipitated with polyethylene glycol (PEG), and concentrated in PBS pH 7.4 + 0.02% sodium azide. Antibody concentration was determined by ELISA.
pals-5