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pH‐Gradient development and focusing speed in thin‐layer polyacrylamide gel isoelectric focusing: A comparison between PharmalyteR, AmpholineR and ServalytR

The rate of pH gradient development and protein migration towards their isoelectric points(pI) were investigated for thin‐layer polyacrylamide gel isoelectric focusing (PAGIF) using Pharmalyte 3–10, Ampholine 3.5–9.5 and Servalyt 2–11 as carrier ampholytes. Using volt‐hours(Vh) as the controlling parameter, it was found that both pH gradient development and protein focusing proceeded very similarly with all three carrier ampholytes, Only two significant (but minor) differences between the carrier ampholytes were observed: (1) due to a faster voltage rise with Pharmalyte, the Vh needed for focusing are achieved faster than with the other two carrier ampholytes and (2) focusing of protein with alkaline pI's (> pH 8) is slightly slower with Servalyt (both in time and Vh). This is presumably explained by the low field strength at the alkaline end of the pH gradient with Servalyt. Also, it was found that pH gradient development was completely independent of applied voltage when Vh were used as controlling parameter.