Determination of Riboflavin Binding Protein (Rfbp) in SDS /Native PAGE ElectrophoresisAuthor(s): M. Bapu Rao, M.Vijay Kumar and G.Rajender
Riboflavin binding protein (Rfbp) was isolated from Hen (Gallus gallus) and Peacock(Pavo cristatus) egg-white, egg-yolk. The protein was purified in two steps, DEAE-Sephadex A-50 ion exchange chromatography. The protein was eluted with phosphate buffer pH 7.3 containing 0.5 M sodium chloride. The final purification of protein was achieved on sephadex G-100. Collected Protein fractions were determined by the method of Lowry. Absorbance was measured at 280 nm and 455 nm using UV-visible spectrophotometer. The purity of the protein was judged on cylindrical and slab gel electrophoresis, SDS (Sodium do-doceyl sulphate)-PAGE (Poly acrylamide gel electrophoresis) technique.Sephadex G-100 fraction Rfbp moved as a single band both on the Slab and Cylindrical gels .Comparison of the mobility of Rfbp with that of the standard molecular weight marker proteins revealed with that the Rfbp had a molecular weight close to 29,000 kilodaltons.Interestingly, hen egg-white Rfbp and peacock egg- white, yolk Rfbp had the same molecular weight as revealed by the SDS polyacrylamide gel electrophoresis.