Hybridisoelectric focusing in rehydrated immobilized pH gradients with added carrier ampholytes: Demonstration of human globins

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Abstract
In contrast to hitherto described procedures, gels containing immobilized pH gradients were prepared from buffered solutions of constant, near neutral pH at ambient temperature. The washed, dried and rehydrated gels were run in the presence or absence of free carrier ampholytes, 8 M urea, Triton X‐100, 2‐mercaptoethanol and dithiothreitol, respectively. The patterns of human globins were compared for linear and modified pH gradients in the range between pH 6 and 10. By adding free carrier ampholytes to the gels the prerun time of urea‐containing gels was reduced from many hours to 60–90 min. The run time with samples was reduced from overnight to 2 h at 3000 V per 10 cm. The amount of sample which had to be applied to obtain clearly visible patterns was found to be close to that used for conventional isoelectric focusing with carrier ampholytes. Upon drying in the laboratory atmosphere an oxidizing activity is generated in polyacrylamide gels which produces artifacts of selected test proteins when separated in the rehydrated gels. The addition of 2% 2‐ mercaptoethanol or 50 mM dithiothreitol to the rehydration solution prevented the formation of these artifacts. Hybrid isoelectric focusing in rehydrated immobilized pH gradients with added carrier ampholytes combines the advantageous properties of isoelectric focusing in immobilized and conventional carrier ampholyte generated pH gradients.