Purification of wheat glutenin subunits by preparative acid and two‐dimensional electrophoresis

Curioni, Andrea; Morel, Marie‐Hélène; Furegon, Luca; Redaelli, R.; Peruffo, A. Dal Belin

doi:10.1002/elps.11501601168

Cited by 8 publications

(5 citation statements)

References 13 publications

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“…Optimization of A-PAGE Preparative Fractionation of Gliadins. According to preliminary observations and in agreement with the literature (Curioni et al, 1995), the preparative system resolution was highly dependent on the separation gel length. It was found that using gels 5 cm in length, a separation of the whole gliadin fraction could be achieved.…”

Section: Resultssupporting

confidence: 88%

“…Regarding the purifica-tion of wheat storage proteins, Curioni et al (1989) described the use of an electroendosmotic preparative electrophoretic method to separate high molecular weight (HMW) glutenins, performing the separation under denaturing conditions at pH 8.3 in the presence of sodium dodecylsulfate (SDS). Curioni et al (1995) described a method for separation of HMW and LMW glutenins using preparative electrophoresis at acid pH.…”

Section: Introductionmentioning

confidence: 99%

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Preparative Fractionation of Gliadins by Electrophoresis at pH 3.1 (A-PAGE)

Rumbo

Chirdo

Giorgieri

et al. 1999

J. Agric. Food Chem.

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Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.

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Section: Resultssupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Preparative Fractionation of Gliadins by Electrophoresis at pH 3.1 (A-PAGE)

Rumbo

Chirdo

Giorgieri

et al. 1999

J. Agric. Food Chem.

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“…Recently, the separation of some reduced, alkylated LMW-GS in a one-step procedure using preparative acid PAGE was described (Curioni et al 1995). This method is attractive as it involves minimal manipulation but uses alkylation of the subunits which alters their functionality in mixing studies.…”

Section: Preparative Electrophoresis and Isoelectric Focusingmentioning

confidence: 99%

Isolation and Functionality Testing of Low Molecular Weight Glutenin Subunits

1998

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Cereal Chem. 75(1):30-36Various protein fractionation techniques have been applied to the isolation and purification of milligram quantities of low molecular weight glutenin subunits (LMW-GS). No single technique was applicable to the purification of the majority of the subunits. Partial purification of certain LMW-GS was obtained using ion-exchange chromatography and reversedphase HPLC. Preparations containing αand γ-type subunit sequences did not strengthen dough when incorporated into a base flour, whereas preparations containing a subunit with an N-terminal methionine residue (METSHIPGL-) did. Using preparative isoelectric focusing over a narrow pH range, it was possible to purify (to ≈90% purity) a B subunit that also had the N-terminal sequence of METSHIPGL-. This polypeptide, when incorporated into a base flour, had a dough strengthening effect in mixing trials, but less so than an equivalent amount of a high molecular weight glutenin subunit.Publication no. C-1998-0105-05R.

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“…There is evidence for the particular mildness of native electrophoresis in the fractionation of photosynthetic complexes (vs. a combination of density-gradient centrifugation with ion-exchange chromatography [298] or solely chromatography [299]). For some proteins, chromatography provides insufficient resolution, while the electrophoretic separation succeeds in a preparative scale [300,301]. Chromatographic, density gradient and electrophoretic approaches have been applied to the preparative isolation of respirasomes (see Section 4.1).…”

Section: Methods Comparison and Limitations Of Preparative Electrophormentioning

confidence: 99%

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Seelert

Krause

2008

Electrophoresis

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Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.

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Purification of wheat glutenin subunits by preparative acid and two‐dimensional electrophoresis

Cited by 8 publications

References 13 publications

Preparative Fractionation of Gliadins by Electrophoresis at pH 3.1 (A-PAGE)

Preparative Fractionation of Gliadins by Electrophoresis at pH 3.1 (A-PAGE)

Isolation and Functionality Testing of Low Molecular Weight Glutenin Subunits

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

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