Westermeier R, Postel W, Weser J, Görg A
J Biochem Biophys Methods. 1983 Dec;8(4):321-30. doi: 10.1016/0165-022x(83)90006-4.
Due to the high reproducibility of pH gradient slope and width, immobilized pH gradients (IPG) have been used as the first dimension of two-dimensional techniques in order to generate maps of constant spot position in the pI/Mr plane. However, when coupling IPG to SDS (sodium dodecyl sulphate) gels two problems were encountered: vertical streaking, due to incomplete zone solubilization in SDS, and horizontal streaking, due to spot fusion along the pH axis caused by the electroendosmosis of the charged Immobiline gels. Two methodical modifications are herewith described to overcome these drawbacks: (a) the SDS equilibration time of the first-dimension gel has been prolonged to at least 30 min; (b) the SDS electrophoresis gel has been cast together with a starting gel, containing 2.5 mM of each Immobiline species used in the first dimension, which serves as a transition from the charged to the uncharged gel.
由于pH梯度斜率和宽度具有较高的重现性，固定化pH梯度（IPG）已被用作二维技术的第一维，以便在pI/Mr平面上生成斑点位置恒定的图谱。然而，当将IPG与SDS（十二烷基硫酸钠）凝胶偶联时，遇到了两个问题：由于SDS中区域溶解不完全导致的垂直条纹，以及由于带电的固定化两性电解质凝胶的电渗作用沿pH轴斑点融合导致的水平条纹。本文描述了两种方法改进以克服这些缺点：（a）将第一维凝胶的SDS平衡时间延长至至少30分钟；（b）将SDS电泳凝胶与起始凝胶一起浇铸，起始凝胶含有在第一维中使用的每种固定化两性电解质2.5 mM，其作为从带电凝胶到不带电凝胶的过渡。
