Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1037, Issue 1-2, Pages 491-499Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2003.11.025
Keywords
reviews; isoelectric points; isoelectric focusing; proteins; amino acids
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Different ways of determining isoelectric points (pI) of proteins in capillary isoelectric focusing are reviewed here. Due to the impossibility of direct pH measurements in the liquid phase, such assessments have to rely on the use of pI markers. Different types of pI markers have been described: dyes, fluorescently labelled peptides, sets of proteins of known pI values. It appears that, perhaps, the best system is a set of 16 synthetic peptides, trimers to hexamers, made, to contain each a Trp residue for easy detection at 280 nm. By a careful blend of acidic (Asp. Glu). mildly basic, with pK around neutrality (His), and basic (Lys, Arg) amino acids, it is possible to obtain a series of pl markers with pI values quite evenly distributed along the pH scale, possessing good buffering capacity and conductivity around their pI values and thus focusing as sharp peaks. Another approach to pI determination is the monitoring of the current during mobilization: this allows, with the aid of known pI markers. to calibrate the system with a pI/current graph. Pitfalls and common errors in pl determinations are reviewed here and guidelines given for minimizing such errors in pI estimation. (C) 2003 Elsevier B.V. All rights reserved.
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