Temperature‐dependent spot positional variability in two‐dimensional polypeptide patterns

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Abstract
The effect of temperature, at which isoelectric focusing with immobilized pH gradients is performed, on spot positions and pattern quality in two‐dimensional (2‐D) Electrophoresis was examined. Increased temperatures revealed improved 2‐D patterns with respect to sample entry, resolution, and background staining. Focusing at 20°C was superior to focusing at 10 and 15°C. Even at 30°C, a pattern of well‐resolved polypeptide spots with a minimum amount of horizontal streaking at the basic end was observed. A computer‐based analysis showed that a substantial proportion of polypeptides assumed altered positions in the 2‐D pattern in relation to temperature. Mobility shifts of polypeptides were more variable on the neutral part than on the acidic or basic end. The mobility shifts were not restricted to one direction for all the spots whose migration was altered. However, for any given spot, the direction was the same with subsequent increments of temperature. The results clearly demonstrate that a defined temperature for first‐dimensional isoelectric focusing is a requirement for the reproducibility of 2‐D Electrophoresis. After elimination of the cathodic drift, a major source of variability in 2‐D patterns, associated with carrier ampholytes, temperature control becomes a critical parameter.