期刊
JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS
卷 68, 期 3, 页码 297-301出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.vascn.2013.07.001
关键词
Kinases; Oxidation; PEG-switch; Thiols; Western blotting
资金
- Medical Research Council
- British Heart Foundation
- Leducq Foundation
- Department of Health via the NIHR cBRC award
- Wellcome Trust [085483/Z/08/Z]
- Wellcome Trust [085483/Z/08/Z] Funding Source: Wellcome Trust
- MRC [G0600785, G0700320] Funding Source: UKRI
- British Heart Foundation [RG/12/12/29872, PG/10/98/28655] Funding Source: researchfish
- Medical Research Council [G0700320, G0600785] Funding Source: researchfish
Introduction: Reversible protein cysteine oxidation is recognised as a pivotal post-transitional modification that transduces physiological as well as pathological signalling. Pharmacological interventions that target specific sources of oxidant formation are currently being trialled to ascertain their potential ability to prevent disease progression. To determine the selectivity of such pharmacological treatments and to indentify new drug targets, a suitable method is required to detect target cysteine oxidation. Method: Using a polyethylene glycol (PEG)-based alkylating agent the reversible oxidation of target proteins can be determined using a novel switch method. After reduction and specific labelling of reversibly oxidised thiols with a 'heavy' PEG-tag, samples are resolved on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, Western blotted and immunostained for protein(s) of interest. A mobility shift in a target protein following PEG-alkylation correlates with the reversible oxidative modification. Results: The oxidation of cAMP-and cGMP-dependent protein kinases was detected using the PEG-switch assay in Langendorff-perfused hearts after hydrogen peroxide was administered. Discussion: The PEG-switch assay is a fast effective semi-quantitative method for measuring target reversible cysteine oxidation in complex protein mixtures derived from tissue or cultured cells. (C) 2013 Elsevier Inc. All rights reserved.
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