Resolution of Gγ and Aγ foetal haemoglobin tetramers in immobilized pH gradients

Cossu, Gianfranco; Righetti, Pier Giorgio

doi:10.1016/s0021-9673(01)96506-9

Cited by 35 publications

(13 citation statements)

References 15 publications

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“…It is generally accepted that the resolution limit, in conventional CA-IEF is DpI = 0.02 to at best 0.01 pH-units [45]. Conversely, the corresponding limit, for immobilized pH gradients, has been set at DpI = 0.001 pH-units [46], and verified in practice via the separation of a neutral point mutant of haemoglobin, the so called Ag vs. Gg, fetal chains differing by an Ala vs. Gly substitution [47]. At the light of the present data, one wonders if even CAs could fare better.…”

Section: Resolution In Acidic Cas: the Dpi Limitmentioning

confidence: 99%

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4–6 intervals

et al. 2006

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For studying the M(r) distribution and number of species in narrow-range (2 pH-unit wide, in the nominal pI 4-6 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted consisting of a focusing step in a liquid phase (Rotofor, yielding 20 fractions) followed by orthogonal CE in both, acidic and basic buffers. As a final step, every other fraction was analyzed by CE-MS. The findings: Ampholine contains 80 different M(r) compounds, in the M(r) interval 203 to 893 Da, for a total of 325 isoforms. Bio-Lyte consists of 66 different M(r) species, in the M(r) range 388 to 835 Da, for a total of 436 isoforms. Servalyt is made of 199 different M(r) compounds, in the M(r) interval 204 to 907 Da, for a total of 1302 isoforms. Pharmalyte pH 4-6.5, comprises 217 amphoteres, in the M(r) range 150 to 1179 Da, for a total of 812 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and <5% "poor" species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value.

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Section: Resolution In Acidic Cas: the Dpi Limitmentioning

confidence: 99%

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4–6 intervals

et al. 2006

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“…Immobilized pH gradients were shown to be capable of resolving HbF from HbF-Sardinia (7,8) in ultra-narrow pH gradients spanning 0.22 pH units over an inter-electrode distance of 10cm. The course of the pH gradient corresponds to 0.022 pH units/cm.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Haemoglobin Variants Using Immobilized pH Gradients

Sinha

Galactéros

Righetti

et al. 1993

Clinical Chemistry and Laboratory Medicine

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Summary:We present a new method for analysis of haemoglobin variants in immobilized pH gradients. The isoelectric fractionation is performed in polyacrylamide gels with copolymerized pH gradients between pH 7.0 and 8.0. The common haemoglobin variants (HbA, HbF, HbS, HbC, HbE and HbA 2 ) are clearly resolved within this pH range. The high resolving power and reproducibility of immobilized pH gradients combined with extremely short separation times make this fractionation technique superior to all electrokinetic procedures used for haemoglobin analysis. At present, isoelectric focusing in immobilized pH gradients is a complementary tool for haemoglobin analysis when classical electrophoretic techniques do not provide sufficient resolution. We hope that in the future this technique will gain the popularity it deserves.

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“…All at once, IPGs allowed this "silent majority" to speak up for themselves, parading their diversity along the electrophoretic track, an "outing" process which came well before the "gay pride day", when finally gays and lesbians could come out in the open. Figures 3 and 4 offer a glimpse at such exquisite separations [102,103]. Together with that, IPGs brought "democracy" for the first time in electrokinetic processes.…”

Section: The Galactic Eighties: Colonizing All the Available Space VImentioning

confidence: 93%

“…The cathode is uppermost. Reprinted from [103], with permission. range, nonlinear IPG, strongly flattened in the crowded region, with a sharp upward turn at alkaline pH values [105]; these ranges are by far the most popular in today 2-D map analyses.…”

Section: The Galactic Eighties: Colonizing All the Available Space VImentioning

confidence: 99%

Bioanalysis: Its past, present, and some future

Righetti¹

2004

Electrophoresis

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An overview of about 100 years of bioanalysis is here disastrously attempted. The beginning of rigorous analytical systems can perhaps be traced back to the building and testing of the analytical ultracentrifuge by Svedberg and the apparatus for moving-boundary electrophoresis of Tiselius, both systems relying on expensive and hard to operate machines. In the sixties, Porath discovered porous beads for the determination of relative molecular mass (Mr) of proteins, based on the principle of molecular sieving. Concomitantly, Svensson and his pupil Vesterberg described a revolutionary principle for fractionating proteins in a nonisocratic environment, based on generation of stable pH gradients in an electric field, a technique that went down to history as isoelectric focusing (IEF). Polyacrylamide gel electrophoresis (PAGE), with the brilliant idea of discontinuous buffers, was brought to the limelight and in 1967, sodium dodecyl sulfate (SDS)-PAGE was described, permitting easy assessment of protein purity and reasonable measurements of Mr values of denatured polypeptide chains. By the mid seventies, another explosive concept was realized: orthogonal combination of two unrelated techniques, based on surface charge and mass fractionation, namely, two-dimensional (2-D) PAGE already in the very first papers by O'Farrell elaborated to its utmost sophistication. The eighties saw the systematic growth of 2-D PAGE, accompanied by systematic efforts to develop instrumentation for large-scale production of 2-D maps and computer evaluation for 2-D map analysis, based on the sophisticated algorithms adopted by astronomers for mapping stars in the sky. Another fundamental innovation in the field of IEF was the discovery of immobilized pH gradients (IPGs) that brought the much needed reproducibility in 2-D maps while allowing exquisite resolution in very narrow pH ranges. The nineties were definitely the decade of capillary zone electrophoresis, with the concomitant concept of automation and miniaturization in electrokinetic methodologies. Also 2-D map analysis witnessed a big revival, thanks to the adoption of IPGs for the first dimension. The enormous progress of mass spectrometry resulted in first reports on the analysis of macromolecules and the building of data bases on gene and protein banks. The third millennium is, perhaps, exasperating the concept of miniaturization at all costs, while not disdaining increasingly larger maps for 2-D analysis of complex protein mixtures.

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Resolution of Gγ and Aγ foetal haemoglobin tetramers in immobilized pH gradients

Cited by 35 publications

References 15 publications

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4–6 intervals

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4–6 intervals

Analysis of Haemoglobin Variants Using Immobilized pH Gradients

Bioanalysis: Its past, present, and some future

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