PMID: 3772023Sep 1, 1986Paper

pH measurements in ultranarrow immobilized pH gradients

Journal of Biochemical and Biophysical Methods
C GelfiP G Righetti

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 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.

References

Apr 21, 1976·Journal of Chromatography·P G Righetti, T Caravaggio
Apr 11, 1977·Journal of Chromatography·O Brenna
Jun 18, 1975·Journal of Chromatography·P G RighettiE Gianazza
Apr 1, 1971·Analytical Biochemistry·R V JosephsonR K Lindorfer

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Citations

Sep 24, 2005·The British Journal of Nutrition·Dagmar FuchsHannelore Daniel
Feb 4, 2010·Expert Review of Proteomics·Tadashi Kondo

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