Atmeh Ragheb F, Massad Tariq T, Kana'an Belal M, Abu-Alrob Adam A
Department of Chemical Sciences, University of Science and Technology, Irbid 22110, Jordan.
Anal Biochem. 2008 Feb 15;373(2):307-12. doi: 10.1016/j.ab.2007.11.012. Epub 2007 Nov 17.
A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.
开发了一种从溶液中的混合物中同时分离和分离生物大分子的直接方法。构建了三个凝胶层，其中包括一个10%聚丙烯酰胺凝胶的中间分离层。该凝胶系统在电浓缩装置中形成，位于底部由透析膜包围的收集室上方。将混合物施加在凝胶层上，生物大分子通过电泳在凝胶系统中迁移，在那里它们被分离成离散的条带并电洗脱到收集室中，而无需拆卸装置。分离出的生物大分子以高纯度和浓缩形式从室中取出，准备进行进一步研究。整个过程都可以进行冷却，并且可以根据要纯化的生物大分子的特性修改运行设置和条件。含有糖蛋白卵清蛋白和牛血清白蛋白单体、二聚体和四聚体的混合物的成分被成功分离为浓缩的高纯度级分，回收率良好，范围为70%至89%。其他蛋白质在十二烷基硫酸钠（SDS）或6 M尿素存在的变性条件下也被成功分离。
