Bossi M L, Bossi O, Gelfi C, Righetti P G
Chair of Biochemistry, Faculty of Pharmacy, University of Milan, Italy.
J Biochem Biophys Methods. 1988 Jun-Jul;16(2-3):171-83. doi: 10.1016/0165-022x(88)90028-0.
A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 microliter each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5 degrees to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, followed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90 degrees and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradients is ensured by using stabilizing density gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).
本文描述了一种基于旋转离心原理在小直径（2毫米）聚丙烯酰胺凝胶棒中浇铸固定化pH梯度的新方法。将玻璃管垂直装满等体积的浓溶液和稀溶液（各250微升），将其滴定至所需pH梯度的极值，然后倾斜2.5度至水平。静置5分钟，使两个弯月面滑动至平衡位置，然后将玻璃管以180转/分钟的速度旋转3分钟，接着通过反转旋转方向再以180转/分钟的速度旋转3分钟。由此产生均匀的线性梯度。然后将旋转平台升高至90度，使凝胶在标准条件下聚合。通过使用低粘度的稳定密度梯度（0 - 5%甘油，粘度/密度的最大比值为1.1）确保形成线性且可重复的pH梯度。
