Immobilized pH gradients and reversed-phase high-performance liquid chromatography: A strategy for characterization of haemoglobin variants with electrophoretic mobility identical to that of Hb A

Rochette, Jacques; Righetti, Pier Giorgio; Bosisio, Adriana Bianchi; Vertongen, Françoise; Schneck, G; Boissel, J. P.; Labie, Dominique; Wajcman, Henri

doi:10.1016/s0021-9673(01)87744-x

Cited by 39 publications

(4 citation statements)

References 16 publications

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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: 94%

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

confidence: 94%

“…The DpI between the two bands was calculated to be 0.01 pH units; unstained gel. Reprinted from [102], with permission. Figure 4.…”

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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“…More discri minative electrophoretic methods, as for example iso electric focusing (IEF) on polyacrylamide gels or immobilized pH gradient (IPG) may be useful to separate the abnormal component from the normal one. Haemoglobin variants with an iso-electric point differing from Hb A by 0.01 pH units may be easily separated by IPG IEF in quantities sufficient for fur ther structural analyses [1,2], Any other abnormal physico-chemical property of the mutated haemoglobin may be used as a basis for its isolation. In order to separate Hb Rainier from Hb A by running an IEF in the deoxyform, we have re cently used the difference in iso-electric point be tween the two quaternary conformations of the haemoglobin molecule in relation with an altered alkaline Bohr effect [3].…”

Section: Strategies For Isolation Of Abnormal Haemoglobinsmentioning

confidence: 99%

Strategy for Structural Characterization of Haemoglobin Variants

Baudin-Chich

Rochette²,

Wajcman³

1987

Acta Haematol

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High-performance liquid chromatography allows one to perform effective and rapid separations on microquantities of material. The miniaturization of the techniques led to new approaches involving iso-electric focusing (IEF) of immobilized pH gradient IEF for haemoglobin preparation, successive enzymic hy-drolyses for isolating abnormal peptides and amino acid composition determination after precolumn derivati-zation

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“…324, 1984). In the acidic chamber (pH 6.8) 364 p1 of 0.2 M Immobiline of pK 7.0 and 236 p1 of 0.2 M Immobiline of pK 3.6 were used; for the basic chamber (pH 7.6) the corresponding amounts were 476 and 96 pl, respectively [19]. The catalysts (8 pl of 40% ammonium persulphate and 6 pl of TEMED per chamber) were added directly to the gradient mixer immediately before filling the gel into the cassette.…”

Section: Immobilized Ph Gradientsmentioning

confidence: 99%

Antenatal diagnosis of β‐thalassemia by isoelectric focusing in immobilized pH gradients

Manca¹,

Cossu²,

Angioni

et al. 1986

American J Hematol

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A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7-7.6 span, HbA and HbFac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6-8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of beta-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -Fac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous beta-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.

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Immobilized pH gradients and reversed-phase high-performance liquid chromatography: A strategy for characterization of haemoglobin variants with electrophoretic mobility identical to that of Hb A

Cited by 39 publications

References 16 publications

Bioanalysis: Its past, present, and some future

Bioanalysis: Its past, present, and some future

Strategy for Structural Characterization of Haemoglobin Variants

Antenatal diagnosis of β‐thalassemia by isoelectric focusing in immobilized pH gradients

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