A procedure for the purification of beta-lactoglobulin from bovine milk using gel filtration chromatography at low pH.
A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4-5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm x 83 cm) of Bio-Gel P10 and has the potential for scaling up.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
Zainab
Z
Naqvi
Rizwan Hasan
RH
Khan
Mohammed
M
Saleemuddin
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000191:economics / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry*
D007782:Lactoglobulins