Isoelectric Focusing with Immobilized pH Gradients

Righetti, Pier Giorgio; Gianazza, Elisabetta; Gelfi, Cecilia

doi:10.1080/03602548708058542

Cited by 7 publications

(6 citation statements)

References 46 publications

(14 reference statements)

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“…The resolving power of immobilized pH gradients is in the magnitude of ΔρΙ of 0.001 pH units (21). We demonstrate that this high resolution is maintained in our system with two known haemoglobin variants.…”

Section: Discussionmentioning

confidence: 72%

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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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Section: Discussionmentioning

confidence: 72%

“…Only two chemicals (acrylamido buffers with pK a of 7.0 and 3.6) are used to generate the pH gradient instead of carrier ampholytes that may contain a multitude of different species (21). This should enable international standardization of the analytical procedure.…”

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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“…For newcomers in the field, the basic, step by step methodology is detailed by Righetti [16]. A I-mm-thick gel was cast on the hydrophilic side of Gel Bond PAG and was made to contain 4 %T, 4 %C solids [ 181.…”

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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“…2/3,1988) and the September 1988 issue of Electrophoresis, both entirely dedicated to IPGs. The basic technology of IPGs has been amply reviewed (15)(16)(17)(18)(19)(20).…”

Section: Introductionmentioning

confidence: 99%