Hybrid isoelectric focusing: Adsorption of proteins onto immobilized pH gradient matrices and desorption by carrier ampholytes

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Abstract
Comparing the separation patterns achieved by hybrid isoelectric focusing in immobilized pH gradients of 1 and 3 meqv/pH/L buffering power, it is demonstrated that a significant proportion of sample protein is lost in gels with the higher buffering power. A buffering power of 1 meqv/pH/L was found to be the lowest acceptable limit for flat bed gel hybrid isoelectric focusing still ensuring adequate control of the immobilized pH gradient. Protein losses were manifested as diminished overall staining intensity, disappearance of individual zones in dilution series, tailing effects and residual material at the application site, all of the above effects being more pronounced for larger than smaller proteins. Adsorption of proteins to charged binding sites of the immobilized pH gradient offers a plausible explanation for the observed phenomena. The visible effects of adsorption could be reduced by the addition of carrier ampholytes, but not by urea or detergents like Triton X‐100, 3‐(3‐chol‐amidopropyl)‐dimethylammonio‐l‐propanesulfonate (CHAPS) or octyl β‐thio‐glucoside, indicating ionic interactions between the immobilized charged groups, carrier ampholytes and proteins. The adsorption phenomena may strongly affect the quantification of proteins in immobilized pH gradients.

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