Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

Izzo, V.; Costa, Maria Assunta; Fiore, Renata Di; Duro, Giovanni; Bellavia, Daniele; Cascone, Eleonora; Colombo, Piergiuseppe; Gioviale, Maria Concetta; Barbieri, Rainer

doi:10.1186/1742-4933-3-7

Cited by 14 publications

(9 citation statements)

References 6 publications

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“…Then, the tubes, caps open, were exposed to UV light for about 30 min, to carry out cross-link between the DNA fragments and the protein(s). The samples were loaded onto a horizontal 12% acrylamide/bisacrylamide gel (29:1 ratio) under non-denaturing conditions and at 300 V (Izzo et al, 2006).…”

Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Naselli

Catanzaro

Bellavia

et al. 2014

Gene

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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5′ flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins.

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Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Naselli

Catanzaro

Bellavia

et al. 2014

Gene

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“…However, this method was poorly reproducible in term of efficient migration of the two proteins, which was attributed to the EEO properties of agarose that might modify the pH of the gel during migration. To overcome this problem, we adapted the apparatus of horizontal PAGE described by Izzo et al [14], and we found that electrophoresis in polyacrylamide gels led not only to results that were more reliable than with agarose, but also improved the resolution of the migration patterns.…”

Section: Discussionmentioning

confidence: 98%

“…Horizontal polyacrylamide gels were cast in a Plexiglas apparatus (15 mm6100 mm61 mm) similar to the one described by Izzo et al [14], except that the wells (6 mm62 mm60.8 mm) were positioned in the middle of the gel, so as to be able to observe the migration of proteins toward both electrodes. The gels were composed of 5% acrylamide (2.7% bisacrylamide), 50 mM Tris pH 8.5, 0.3% ammonium persulfate, 0.1% TEMED, and a suitable detergent.…”

Section: Methodsmentioning

confidence: 99%

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE

Choul-Li¹,

Adams²,

Pattus³

et al. 2008

Electrophoresis

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Horizontal nondenaturing electrophoresis of proteins in polyacrylamide gels was used to observe specific interactions between membrane proteins. The method was particularly well suited for solubilized transporters of the outer membrane of Gram-negative bacteria, and allowed specific complexes of transporter and the inner-membrane protein TonB to be isolated. We have used this method to investigate the interactions between four different outer-membrane transporters, and the TonB proteins from two different organisms. The results show that a stable complex can be isolated on gels for all the proteins studied, but can depend in some cases of the detergent used for solubilization. Furthermore, we observe cross-species interaction as TonB from a given organism can interact with transporters from another organism.

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“…Before developing our HT-PAGE, we noticed that Barbieri et al reported the development of a horizontal sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique that could be applied to analyze both protein and DNA samples. However, the authors did not consider to develop it into a high-throughput technique (Izzo et al, 2006). We thus prepared a horizontal polyacrylamide gel between an acrylic plate that contains one row of 15 protruding teeth of 3 x 1 x 1 mm (particularly made for us by the supplier, from Taobao.com) and a microscopic glass slide (75 x 25 mm) using tris-borate (89 mM) as the gel buffer, as adapted from their report (Izzo et al, 2006).…”

Section: Page) Techniquementioning

confidence: 99%

“…However, the authors did not consider to develop it into a high-throughput technique (Izzo et al, 2006). We thus prepared a horizontal polyacrylamide gel between an acrylic plate that contains one row of 15 protruding teeth of 3 x 1 x 1 mm (particularly made for us by the supplier, from Taobao.com) and a microscopic glass slide (75 x 25 mm) using tris-borate (89 mM) as the gel buffer, as adapted from their report (Izzo et al, 2006). We tested this system by performing an electrophoresis and blotting analysis on the photocrosslinked products of 12 of the Bpa variants of the ε-subunit.…”

Section: Page) Techniquementioning

confidence: 99%

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the “inserted” state of the ATP synthase ε-subunit

Liu

Wang

et al. 2019

Preprint

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ATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of α3β3γδεab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (α3β3γδε) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the ε-subunit could adopt either an "inserted" or "non-inserted" state (with or without interacting with the α/β-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an "inserted" state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimentalconditions. These studies enabled us to uncover that the "inserted" and "non-inserted" states of the ε-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a § low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

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Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

Cited by 14 publications

References 6 publications

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the “inserted” state of the ATP synthase ε-subunit

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