Wu J, Li S C, Watson A
Convergent Bioscience Ltd., Toronto, ON, Canada.
J Chromatogr A. 1998 Aug 21;817(1-2):163-71. doi: 10.1016/s0021-9673(98)00326-4.
Separation conditions for antibodies, glycoproteins and peptides were optimized to fully realize the potential of automated imaged capillary isoelectric focusing (imaged cIEF) for protein analysis. Two commercially available capillary coatings, polyacrylamide and fluorocarbon, were found to provide reproducible results for cIEF separations. Both coatings could last more than 100 runs under normal cIEF conditions. Up to 30 mM salts (Na+) could be added to samples to prevent protein precipitation before and during isoelectric focusing performed under imaged cIEF. Short analysis time of the imaged cIEF also aided in the prevention of protein precipitation. High current at the beginning of the focusing for samples in salt could be avoided by applying a voltage gradient. Additions of up to 6 M urea and 20% glycerol could enhance solubility of proteins and peptide. Imaged cIEF was applied to the quantitation of monoclonal antibodies.
优化了抗体、糖蛋白和肽的分离条件，以充分发挥自动成像毛细管等电聚焦（成像cIEF）在蛋白质分析方面的潜力。发现两种市售的毛细管涂层，聚丙烯酰胺和碳氟化合物，可为cIEF分离提供可重复的结果。在正常cIEF条件下，两种涂层都可以持续运行100多次。在成像cIEF进行等电聚焦之前和期间，可向样品中添加高达30 mM的盐（Na+）以防止蛋白质沉淀。成像cIEF的短分析时间也有助于防止蛋白质沉淀。通过施加电压梯度可以避免盐溶液中样品聚焦开始时的高电流。添加高达6 M的尿素和20%的甘油可以提高蛋白质和肽的溶解度。成像cIEF被应用于单克隆抗体的定量分析。
