Gelfi C, Morelli A, Rovida E, Righetti P G
J Biochem Biophys Methods. 1986 Sep;13(2):113-24. doi: 10.1016/0165-022x(86)90023-0.
It is possible to measure pH values in immobilized pH gradients (IPG) when the polyacrylamide matrix is made to contain an additional, carrier ampholyte-generated pH gradient. After an IPG run, 5 mm gel segments, along the separation axis, are cut and eluted in 300 microliter of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 microliter) are eluted in 900 microliter of 100 mM KCl and the pH assessed with a differential pH meter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert delta pH values into an actual pH curve. The reproducibility of the system is better than +/- 0.05 pH units, with a ca. 15% error over a 0.3 pH unit span. In ultranarrow pH gradients, it is imperative to use mixtures of all commercially available carrier ampholytes, so as to smoothen conductivity and buffering capacity gaps. By the present method, it is also possible to convert a wide (2-3 pH unit) carrier ampholyte interval into a narrow (0.2-0.3 pH unit) one.
当聚丙烯酰胺基质含有额外的、由载体两性电解质产生的pH梯度时，就可以在固定化pH梯度（IPG）中测量pH值。IPG运行后，沿分离轴切割5毫米的凝胶片段，在300微升10 mM KCl中洗脱，并用标准pH计读取pH值。使用超窄pH梯度时，将较大的凝胶片段（约265微升）在900微升100 mM KCl中洗脱，并用差分pH计评估pH值。在后一种情况下，使用内部或外部标准作为参考或起点，将ΔpH值转换为实际的pH曲线。该系统的重现性优于±0.05 pH单位，在0.3 pH单位范围内误差约为15%。在超窄pH梯度中，必须使用所有市售载体两性电解质的混合物，以平滑电导率和缓冲容量的差距。通过本方法，还可以将宽（2 - 3 pH单位）的载体两性电解质间隔转换为窄（0.2 - 0.3 pH单位）的间隔。
