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.

Endoplasmic reticulum stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of Arabidopsis thaliana

“…After incubation, the gels were washed with distilled water twice and incubated for 5min in staining solution containing 1% FeCl 3 and 1% K 3 Fe(CN) 6 . GR isoforms were detected using 7.5% separating gels according to Hou et al (2004). GR isoforms were detected by incubating the gels in a solution containing 10mM TRIS-HCl (pH 7.9), 4mM GSSG, 1.5mM NADPH, and 2mM DTNB for 20min.…”

Acute Liver Failure in Rats Activates Glutamine-Glutamate Cycle but Declines Antioxidant Enzymes to Induce Oxidative Stress in Cerebral Cortex and Cerebellum

“…[22] with some modifications. After 10% native PAGE of 60 µg protein in each lane, gels were incubated with 50 mM Tris-HCl (pH 7.9) containing 4 mM GSSG, 1.5 mM NADPH and 2 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) for 20 min.…”

Activation of Neuronal Nitric Oxide Synthase in Cerebellum of Chronic Hepatic Encephalopathy Rats is Associated with Up-regulation of NADPH-Producing Pathway

“…Level of active GR was determined following the method of Hou et al [42] with some modification. After 10% native PAGE of 60-μg protein in each lane, gels were incubated with (in mM) 50 Tris-HCl (pH 7.9) containing 4.0 oxidized GSH (GSSG), 1.5 NADPH and 2,5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min.…”