PH measurements in ultranarrow immobilized pH gradients

Abstract

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 μl of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 μl) are eluted in 900 μl of 100 mM KCl and the pH assessed with a differential pHmeter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert Δ 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.