Abstract

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2′,5′-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.

Original languageEnglish
Pages (from-to)2926-2931
Number of pages6
JournalElectrophoresis
Volume25
Issue number17
DOIs
Publication statusPublished - Sep 2004

Fingerprint

Glutathione Reductase
Electrophoresis
Sodium Dodecyl Sulfate
Gels
Yeast
Spinacia oleracea
Polyacrylamide Gel Electrophoresis
Glutathione Disulfide
Yeasts
Purification
Methylphenazonium Methosulfate
Nitrobenzoates
Dithionitrobenzoic Acid
Native Polyacrylamide Gel Electrophoresis
Negative Staining
Tromethamine
2-Propanol
NADP
Washing
Saccharomyces cerevisiae

Keywords

  • Glutathione reductase
  • Native polyacrylamide gel electrophoresis
  • Polyacrylamide
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels. / Hou, Wen Chi; Liang, Hong Jen; Wang, Ching Chiung; Liu, Der Zen.

In: Electrophoresis, Vol. 25, No. 17, 09.2004, p. 2926-2931.

Research output: Contribution to journalArticle

@article{f247ff1fd3c740ecae2f023e6e61fe8c,
title = "Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels",
abstract = "Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5{\%} native polyacrylamide gel electrophoresis (PAGE) gels or 15{\%} sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2′,5′-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25{\%} isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.",
keywords = "Glutathione reductase, Native polyacrylamide gel electrophoresis, Polyacrylamide, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis",
author = "Hou, {Wen Chi} and Liang, {Hong Jen} and Wang, {Ching Chiung} and Liu, {Der Zen}",
year = "2004",
month = "9",
doi = "10.1002/elps.200406041",
language = "English",
volume = "25",
pages = "2926--2931",
journal = "Electrophoresis",
issn = "0173-0835",
publisher = "Wiley-VCH Verlag",
number = "17",

}

TY - JOUR

T1 - Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels

AU - Hou, Wen Chi

AU - Liang, Hong Jen

AU - Wang, Ching Chiung

AU - Liu, Der Zen

PY - 2004/9

Y1 - 2004/9

N2 - Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2′,5′-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.

AB - Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2′,5′-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.

KW - Glutathione reductase

KW - Native polyacrylamide gel electrophoresis

KW - Polyacrylamide

KW - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

UR - http://www.scopus.com/inward/record.url?scp=4644295671&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=4644295671&partnerID=8YFLogxK

U2 - 10.1002/elps.200406041

DO - 10.1002/elps.200406041

M3 - Article

C2 - 15349931

AN - SCOPUS:4644295671

VL - 25

SP - 2926

EP - 2931

JO - Electrophoresis

JF - Electrophoresis

SN - 0173-0835

IS - 17

ER -