Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
Prep Biochem Biotechnol. 2010;40(4):326-36. doi: 10.1080/10826068.2010.525405.
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.
从牛乳中提取β-乳球蛋白（β-LG）的一种简单快速的方法。该方法利用了β-LG 和其他乳清成分在分子量上的显著差异，以及前者在酸性 pH 下呈单体形式存在的事实。在 pH 3.0 下，使用 Bio-Gel P10 柱进行乳清凝胶过滤。残留的酪蛋白和其他乳蛋白被排除在凝胶之外，β-LG 和α-乳白蛋白（α-LA）则呈现出两个完全分离的峰。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）表明β-LG 已被纯化到明显的均一状态，而吸收、荧光和圆二色性光谱表明该蛋白具有天然构象。Western blot 分析表明，用纯化的β-LG 免疫兔子产生的抗体也能与商业牛蛋白发生反应。该方法仅需 4-5 小时，即可从小型 Bio-Gel P10 柱（2.3 cm x 83 cm）中纯化约 10mg 的β-LG，具有扩大规模的潜力。
