pH measurements after isoelectric focusing in immobilized pH gradients

It is impossible to measure pH values in immobilized pH gradients either with a surface electrode or by cutting gel slices and eluting in 10 mM potassium chloride solution. The use of reversible gels, cross-linked with bisacrylylcistamine, improves the measurements in the acidic region, but gives false values in the alkaline region, owing to the buffering power of added 2-mercaptoethanol or dithiothreitol. By using mixed-type gels, containing Immobilines and 1% carrier ampholytes, accurate and reliable pH measurements can be obtained. The discrepancy between the theoretical slope of the immobilized pH gradient and the actual pH values obtained by reading the pH of eluted Ampholine focusing in the same gel fragment is less than 0.1 pH unit over a 1 pH unit span. The effects of temperature and of carbon dioxide absorptionbnd on pH readings are demonstrated and evaluated.