Zakharov S F, Kwok S H, Sokoloff H, Chang H T, Radko S P, Chrambach A
Section on Macromolecular Analysis, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
Electrophoresis. 1998 Jul;19(10):1625-30. doi: 10.1002/elps.1150191018.
An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.
一种最近在市场上可买到的自动凝胶电泳仪，能让人在电泳过程中实时跟踪条带，因此有助于评估带宽与迁移时间的函数关系（分散系数）、分辨率和条带形状。这些测定假定条带面积在迁移时间和不同凝胶浓度下保持恒定。本研究的目的是验证这些假设。结果发现，无论是天然荧光的还是经荧光素羧酸盐标记的代表性蛋白质和十二烷基硫酸钠（SDS）-蛋白质，在琼脂糖和聚丙烯酰胺凝胶电泳中，只要满足以下条件，其条带面积随迁移时间的变化接近恒定：（i）条带下方的蛋白质浓度足够低，以避免荧光的自猝灭；（ii）在测量条带面积的时间段内，目标蛋白质与污染物的分离进展充分；（iii）峰下基线定义良好。然而，条带面积会随着凝胶浓度的增加而减小。蛋白质峰呈现前沿峰和拖尾峰。在不同迁移时间下，拖尾峰总面积与总面积的比值似乎接近恒定。然而，该比值会随着凝胶浓度的增加而增大。拖尾峰面积似乎不是荧光检测的假象，因为在对电泳后从凝胶切片中洗脱的蛋白质进行荧光分析时会重现这一现象。然而，这可能是由于在重复荧光峰检测条件下的光化学破坏所致。
