Shang Tanya Q, Ginter Joy M, Johnston Murray V, Larsen Barbara S, McEwen Charles N
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA.
Electrophoresis. 2003 Jul;24(14):2359-68. doi: 10.1002/elps.200305502.
The field of proteomics requires methods that offer high sensitivity and wide dynamic range. One of the strategies used to improve the dynamic range is sample prefractionation, such as microsolution isoelectric focusing (IEF). We have modified a commercial solution IEF instrument, the Rotofor, to prefractionate protein mixtures by carrier ampholyte-free solution IEF. The focusing chamber of the Rotofor was divided into several compartments by polyacrylamide membranes with imbedded Immobiline mixtures of specific pH values. When an electric field is applied, each protein migrates to the compartment confined by membranes with pH values flanking its isoelectric point. The approach was demonstrated for the focusing of myoglobin into a predicted compartment, as well as the separation of a complex soluble yeast protein mixture into several distinct fractions. The proteins were dissolved in water or 30% isopropanol. The method is applicable to both gel-based and solution-phase protein identification methods, without the need for further sample preparation.
蛋白质组学领域需要具备高灵敏度和宽动态范围的方法。用于扩大动态范围的策略之一是样品预分级，例如微溶液等电聚焦（IEF）。我们对商用溶液IEF仪器Rotofor进行了改进，通过无载体两性电解质溶液IEF对蛋白质混合物进行预分级。Rotofor的聚焦室通过嵌入特定pH值的固定化电解质混合物的聚丙烯酰胺膜被分成几个隔室。施加电场时，每种蛋白质会迁移到由pH值与其等电点相邻的膜所限定的隔室中。该方法已被证明可将肌红蛋白聚焦到预测的隔室中，以及将复杂的可溶性酵母蛋白质混合物分离成几个不同的级分。蛋白质溶解于水或30%异丙醇中。该方法适用于基于凝胶和溶液相的蛋白质鉴定方法，无需进一步的样品制备。
