Binding analysis of bovine milk proteins, especially casein interactions and the interaction between α-casein and lactoferrin, using beads immobilised with zinc ion, poly-l-lysine or α-casein

Binding analysis of horse and bovine ferritins using beads immobilized with alpha-casein-: detection of ferritin binding or nonbinding with heme using alpha-casein-immobilized beads

“…CasB has a possibility to detect heme-binding ferritin because Cas binds bovine ferritin but not its apoferritin, which lacks iron and heme, by hememediated binding (8,17). Although PL binds heme (9), horse spleen ferritin and its apoferritin form bind to PLB different from Cas, suggesting that PLB binds ferritin directly, likely through hydrophobic interactions, similar to the binding of PLB and bovine lactoferrin (10). On the other hand, CasB partly bound horse ferritin but did not bind the apoferritin form.…”

“…CasB was prepared using CNBr-activated Sepharose 4B according to the method described previously (10).…”

“…After washing, 100 µl of ALP-conjugated NeutrAvidin (1 µg/ml) diluted in 0.1% (w/v) BA was added to each well, and the plate was incubated at 37°C for 1.5 hr. After washing, the ALP enzyme reaction was carried out using disodium p-nitrophenyl phosphate as previously described (10). The concentration of p-nitrophenol produced by the ALP reaction was determined by measuring the absorbance at 405 nm.…”

“…In bead methods, metal-binding protein and protein-protein interactions can be studied using beads with immobilized metal ions, brinogen, α-casein (CasB) or PL (PLB) (9,10). This study, rstly, conducted the binding analysis with PLB using commercial available horse spleen ferritin and it's apoferritin, but these bindings are independent on the iron because PLB bound apoferritin as well as holoferritin different from CasB only binding with holoferritin.…”