SDS-PAGE Focusing:  Preparative Aspects

Zilberstein, Gleb; Korol, Leonid; Righetti, Pier Giorgio; Bukshpan, S.

doi:10.1021/ac701598y

Cited by 14 publications

(15 citation statements)

References 30 publications

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“…Standard SDS‐PAGE was used to evaluate proteins separation via the FFE instrument . For an SDS‐PAGE run, 375 mM (pH 8.8) Tris‐HCl solution (375 mM Tris was titrated by HCl to pH 8.8) and 125 mM (pH 6.8) Tris‐HCl solution (125 mM Tris was titrated by HCl to pH 6.8) were prepared as the separation buffer and stacking buffer, respectively.…”

Section: Methodsmentioning

confidence: 99%

Experimental study on the optimization of general conditions for a free‐flow electrophoresis device with a thermoelectric cooler†

Yan

Yang

Zhang

et al. 2014

J of Separation Science

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With a given free-flow electrophoresis device, reasonable conditions (electric field strength, carrier buffer conductivity, and flow rate) are crucial for an optimized separation. However, there has been no experimental study on how to choose reasonable general conditions for a free-flow electrophoresis device with a thermoelectric cooler in view of Joule heat generation. Herein, comparative experiments were carried out to propose the selection procedure of general conditions in this study. The experimental results demonstrated that appropriate conditions were (i) <67 V/cm electric field strength; (ii) lower than 1.3 mS/cm carrier buffer conductivity (Tris-HCl: 20 mM Tris was titrated by HCl to pH 8.0); and (iii) higher than 3.6 mL/min carrier buffer flow rate. Furthermore, under inappropriate conditions (e.g. 400 V voltage and 40 mM Tris-HCl carrier buffer), the free-flow electrophoresis separation would be destroyed by bubbles caused by more Joule heating. Additionally, a series of applications under the appropriate conditions were performed with samples of model dyes, proteins (bovine serum albumin, myoglobin, and cytochrome c), and cells (Escherichia coli, Streptococcus thermophilus, and Saccharomyces cerevisiae). The separation results showed that under the appropriate conditions, separation efficiency was obviously better than that in the previous experiments with randomly or empirically selected conditions.

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Section: Methodsmentioning

confidence: 99%

Experimental study on the optimization of general conditions for a free‐flow electrophoresis device with a thermoelectric cooler†

Yan

Yang

Zhang

et al. 2014

J of Separation Science

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show abstract

“…This first report was soon followed by a second one, in which it was proved that the same method could be exploited also for small‐scale preparative purposes, although in a more complex experimental set‐up, considering that these runs were performed not in a firm gel, but in a viscous polymer solution, in the absence of cross‐linkers 27. Minimization of migration of the liquid cationic polymers was obtained via use of low voltage gradients and by arranging for a buffer conductivity gradient along the migration path.…”

Section: The Third Generation Of Ief: Gel Matrices With Immobilizedmentioning

confidence: 99%

Third generation of focusing: Gel matrices with immobilized cation gradients

Zilberstein¹,

Bukshpan²,

Righetti

2010

Electrophoresis

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A novel method is reviewed here for separation of polyanions, based not on conventional zone electrophoretic means, but on a "steady-state" process by which said polyanions are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix. As the total negative surface charge of such polyanions matches the surrounding charge density of the matrix, they stop migrating and remain stationary, as typical of steady-state separation techniques. This technique has been successfully applied to SDS-protein micelles, DNAs, RNAs and heparins, with remarkable separations, often much superior than those obtained in conventional techniques. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced charge densities. This technique resembles a classical IEF process, with the proviso that the polyanions cannot be applied at any position along the gel matrix, but only at the point of low (or zero) charge density. The merits and limits of the technique are assessed.

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“…In 2007 SDS-PAGE focusing was introduced [349]. This method may also be useful for preparative electrophoresis [350]. Ultimately, the main issue will be adaptation of the electrokinetic method to a given separation problem.…”

Section: Future Perspectives and Conclusionmentioning

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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SDS-PAGE Focusing: Preparative Aspects

Cited by 14 publications

References 30 publications

Experimental study on the optimization of general conditions for a free‐flow electrophoresis device with a thermoelectric cooler†

Experimental study on the optimization of general conditions for a free‐flow electrophoresis device with a thermoelectric cooler†

Third generation of focusing: Gel matrices with immobilized cation gradients

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

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