A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3- naphthalenedicarboxaldehyde
-
Altmetric Citations
Description
This study reports the development of a new assay for the rapid determination of protein glutathionylation in tissues and cell lines using commercially available reagents and standard instrumentation. In this method cells are homogenized in the presence of N-ethylmaleimide to eliminate free thiols and the proteins are precipitated with acetone. Subsequently, the disulfide-bound glutathione is eluted from the protein by the addition of tris(2-carboxyethyl)phosphine and reacted with...[Show more]
dc.contributor.author | Menon, Deepthi | |
---|---|---|
dc.contributor.author | Board, Philip | |
dc.date.accessioned | 2015-12-10T22:30:38Z | |
dc.identifier.issn | 0003-2697 | |
dc.identifier.uri | http://hdl.handle.net/1885/55180 | |
dc.description.abstract | This study reports the development of a new assay for the rapid determination of protein glutathionylation in tissues and cell lines using commercially available reagents and standard instrumentation. In this method cells are homogenized in the presence of N-ethylmaleimide to eliminate free thiols and the proteins are precipitated with acetone. Subsequently, the disulfide-bound glutathione is eluted from the protein by the addition of tris(2-carboxyethyl)phosphine and reacted with 2,3-napthalenedicarboxaldehyde to generate a highly fluorescent product. Lymphoblastoid cell lines were found to have glutathionylation levels in the range of 0.3-3 nmol/mg protein, which were significantly elevated after treatment of the cells with S-nitrosoglutathione. Mouse tissues including liver, kidney, lung, heart, brain, spleen, and testes were found to have glutathionylation levels between 1 and 2.5 nmol/mg protein and the levels tended to increase after treatment of mice with doxorubicin. In contrast, mouse skeletal muscle glutathionylation was significantly higher (4.2 ± 0.33 nmol/mg, p < 0.001) than in other tissues in untreated mice and decreased to 1.9 ± 0.15 nmol/mg after doxorubicin treatment. This new method allows rapid measurement of cellular glutathionylation in a high-throughput 96-well plate format. | |
dc.publisher | Academic Press | |
dc.source | Analytical Biochemistry | |
dc.subject | Keywords: 2,3 naphthalenedicarboxaldehyde; acetone; doxorubicin; glutathione; glutathione derivative; n ethylmaleimide; s nitrosoglutathione; tris(2 carboxyethyl)phosphine; animal cell; animal experiment; animal tissue; article; brain; chemical reaction; controlled Detection of glutathionylation with 2,3-naphthalenedicarboxaldehyde; Glutathionylation; Quantification of glutathionylation | |
dc.title | A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3- naphthalenedicarboxaldehyde | |
dc.type | Journal article | |
local.description.notes | Imported from ARIES | |
local.identifier.citationvolume | 433 | |
dc.date.issued | 2013 | |
local.identifier.absfor | 060101 - Analytical Biochemistry | |
local.identifier.ariespublication | u4020362xPUB321 | |
local.type.status | Published Version | |
local.contributor.affiliation | Menon, Deepthi, College of Medicine, Biology and Environment, ANU | |
local.contributor.affiliation | Board, Philip, College of Medicine, Biology and Environment, ANU | |
local.description.embargo | 2037-12-31 | |
local.bibliographicCitation.issue | 2 | |
local.bibliographicCitation.startpage | 132 | |
local.bibliographicCitation.lastpage | 136 | |
local.identifier.doi | 10.1016/j.ab.2012.10.009 | |
local.identifier.absseo | 920102 - Cancer and Related Disorders | |
dc.date.updated | 2016-02-24T10:27:55Z | |
local.identifier.scopusID | 2-s2.0-84870653940 | |
local.identifier.thomsonID | 000314009300010 | |
Collections | ANU Research Publications |
Download
File | Description | Size | Format | Image |
---|---|---|---|---|
01_Menon_A_fluorometric_method_to_2013.pdf | 367.36 kB | Adobe PDF | Request a copy |
Items in Open Research are protected by copyright, with all rights reserved, unless otherwise indicated.
Updated: 17 November 2022/ Responsible Officer: University Librarian/ Page Contact: Library Systems & Web Coordinator