Micropreparative one‐ and two‐dimensional electrophoresis: Improvement with new photopolymerization systems

Rabilloud, Thierry; Vincon, Mathilde; Garin, Jérôme

doi:10.1002/elps.11501601234

Cited by 25 publications

(26 citation statements)

References 19 publications

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“…A slight modification to the photopolymerisation method previously reported [17] was used. Aqueous solutions of the photoinitiators RBF (20 mM), STS (250 mM) and DPIC (10 mM) or RBF (20 mM) and TEMED (0.5±2 mL) were added to each monomer solution.…”

Section: Photopolymerisationmentioning

confidence: 99%

On the mechanism of background silver staining during sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Patras

Qiao

1999

Electrophoresis

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A novel experimental procedure to study the factors that contribute to background silver staining in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) is described. A mechanism is proposed for background silver staining, which was found to result primarily from the redox initiator system using ammonium persulphate (APS) and an amine such as N,N,N′,N′‐tetramethylethylenediamine (TEMED).

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Section: Photopolymerisationmentioning

confidence: 99%

On the mechanism of background silver staining during sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Patras

Qiao

1999

Electrophoresis

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“…The initial separation in many proteomics protocols utilizes polyacrylamide gel electrophoresis as the gels are easily and rapidly prepared and possess the necessary property of high resolution [5,6]. An SDS polyacrylamide gel is widely used as the second dimensional-denatured gel when complex protein mixtures need to be separated.…”

Section: Introductionmentioning

confidence: 99%

Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate‐gel electrophoresis

Sun

Anderson

2004

Electrophoresis

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Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochrome c was electrophoresed on polyacrylamide gels and subjected to trypsin in-gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochrome c isolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.

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“…For the identification of oxidatively modified residues, the D1 and D2 proteins were resolved on 12.5-20% acrylamide lithium dodecyl sulfate-PAGE gradient gels by using a nonoxidizing system as was described (17,45,46). This nonoxidizing gel system exhibited significantly lower levels of artifactual protein oxidation (see fig.…”

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confidence: 99%

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

Kale

Hebert

Frankel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

134

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The Photosystem II reaction center is vulnerable to photoinhibition. The D1 and D2 proteins, lying at the core of the photosystem, are susceptible to oxidative modification by reactive oxygen species that are formed by the photosystem during illumination. Using spin probes and EPR spectroscopy, we have determined that both O 2 . The identification of specific amino acid residues oxidized by reactive oxygen species provides insights into the mechanism of damage to the D1 and D2 proteins under light stress.photosynthesis | Photosystem II | reactive oxygen species | photo inhibition | mass spectrometry P hotosystem II (PSII) functions as a water-plastoquinone oxidoreductase (1, 2) and is a thylakoid membrane pigmentprotein complex present in all oxygenic photosynthetic organisms (cyanobacteria, algae, and higher plants). Current high-resolution structures of thermophilic cyanobacterial PSII (3, 4), and lower resolution structures of the red algal (5) and higher plant photosystems (6), have been critically important in furthering our understanding of the molecular organization of PSII. Structurally, the PSII reaction center core is composed of five proteins: D1, D2, the α-and β-subunits of cytochrome b 559 , and PsbI. These components bind all of the redox-active cofactors of PSII.Excitation energy transfer and electron transport within PSII are unavoidably associated with production of reactive oxygen species (ROS) when the absorption of light by the chlorophyll antenna exceeds the capacity for energy utilization. Many mechanisms for ROS production have been proposed (for reviews, see refs. 7 and 8). Briefly, singlet oxygen ( 1 O 2 ) may be formed by excitation energy transfer from triplet chlorophylls (formed either by the change in orientation of the spin of an excited electron in the PSII antenna complex, or via charge recombination of the primary radical pair 3 [P 680 •+ Pheo •− ]) to O 2 (9, 10). ROS production by electron transport involves either the two-electron oxidation of water or the one-electron reduction of O 2 on the PSII electron donor and acceptor sides, respectively. On the PSII electron donor side, a twoelectron oxidation of water leads to the formation of hydrogen peroxide (H 2 O 2 ), which may be oxidized to the superoxide anion radical (O 2•−

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Micropreparative one‐ and two‐dimensional electrophoresis: Improvement with new photopolymerization systems

Cited by 25 publications

References 19 publications

On the mechanism of background silver staining during sodium dodecyl sulphate-polyacrylamide gel electrophoresis

On the mechanism of background silver staining during sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate‐gel electrophoresis

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

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