Griess G A, Rogers E, Serwer P
Department of Biochemistry, The University of Texas Health Science Center, San Antonio 78284-7760, USA.
Electrophoresis. 2001 Apr;22(6):981-9. doi: 10.1002/1522-2683()22:6<981::AID-ELPS981>3.0.CO;2-X.
Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R > or = 95 nm) spheres (Serwer, P, Griess, G.A., Anal. Chim. Acta 1998, 372, 299-306). The electrical oscillations are the following electrical field pulses: high field --> low field --> high field, etc. The field is inverted after each pulse; the time-integral of the field can be zero. Response to the ratchet is caused by steric trapping in the high field-direction, but not in the low field-direction. Trapping and, therefore, response to the ratchet decrease as R decreases. The smaller spheres do not respond to the ratchet. In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet. To achieve this objective, the heterogeneity of pore size is increased for the gel used. The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature. If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field-direction, when the above-described pulsed field is biased in the high field-direction. If a particle does respond to the improved ratchet, the particle has a net migration in the low field-direction. Here, the R of ratchet-responding spheres is reduced to 30-50 nm. These ratchet-responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres. The smaller ratchet-responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude. A ratchet-based procedure is developed here to achieve continuous preparative gel electrophoresis.
通过凝胶电泳棘轮进行分级分离先前已成功应用于相对较大（半径R≥95纳米）的球体（Serwer, P, Griess, G.A., 《分析化学学报》1998年，372卷，299 - 306页）。电振荡是以下电场脉冲：高场→低场→高场等。每个脉冲后电场反转；电场的时间积分可以为零。对棘轮的响应是由在高场方向的空间捕获引起的，而在低场方向则不会。捕获以及因此对棘轮的响应随着R的减小而降低。较小的球体对棘轮没有响应。在本研究中，首次使半径R值小于95纳米的球体对类似的凝胶电泳棘轮产生响应。为实现这一目标，所用凝胶的孔径异质性增加。孔径异质性通过以下方式增加：（i）用降解的羟乙基琼脂糖形成凝胶，以及（ii）在相对较高的温度下凝胶化。如果一个颗粒仍然对棘轮没有响应（因为颗粒太小），当上述脉冲电场在高场方向偏置时，该颗粒在高场方向有净迁移。如果一个颗粒确实对改进的棘轮有响应，该颗粒在低场方向有净迁移。在此，对棘轮有响应的球体的R减小到30 - 50纳米。这些对棘轮有响应的球体包括完整的噬菌体颗粒（R = 30纳米）和乳胶球体。较小的对棘轮有响应的球体具有电泳迁移率，其大小随着电场大小的增加而减小。在此开发了一种基于棘轮的方法来实现连续制备凝胶电泳。
