Gel electrofocusing in a natural pH gradient of pH 3–10 generated by a 47‐component buffer mixture

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Abstract
A natural pH gradient with a pH range of 3–10 was generated from a mixture of 47 buffers utilizing a polyacrylamide gel slab format. The gradient is stable for at least 18 h at 100 V/cm of gel, with the attainment of the steady state at 1 h and of isoelectric protein positions within 2.5 h. Gels containing buffer constituents at a final concentration of 10–20 mM exhibit a conductivity which is approximately one order of magnitude less than that exhibited by any synthetic carrier ampholyte mixture (SCAM), thereby permitting a high degree of resolution in analytic applications. Excessive Joule heating and artefactual protein‐protein interaction, both potential drawbacks to electrofocusing at lower ionic strengths, were not observed in this study. The pH gradient generated by the buffer mixture makes possible for the first time electrofocusing of proteins under constant conditions in longitudinal studies, independently of the source and batch of carrier constituents. Furthermore, this system completely circumvents the physical, chemical and biologic problems frequently encountered with SCAMs.