Method for assaying protein nitrosylation
AssigneeThe Johns Hopkins University
InventorsSolomon H. Snyder, Samie R. Jaffrey, Christopher D. Ferris, Hediye Erdjument-Bromage, Paul Tempst
Life of this patent
Estimating the term…
Abstract
Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. A method to detect proteins which contain nitrosothiols involves several steps. Nitrosylated cysteines are converted to tagged cysteines. Tagged proteins can then be detected, for example, by immunoblotting and/or can be purified by affinity chromatography. The method is applicable to the detection of S-nitrosylated proteins in cell lysates following in vitro S-nitrosylation, as well as to the detection of endogenous S-nitrosothiols in selected protein substrates.
Claims35
A method for assaying nitrosylation of protein substrates, comprising:
- treating a test sample comprising at least one protein substrate with an alkyithiolating agent to block free thiol groups on the protein substrate;
- reducing nitrosothiol bonds on the protein substrate to form new free thiol groups;
- removing alkyithiolating agent from the test sample;
- reacting the new free thiol groups on the protein substrate with a detectably tagged, activated mixed disulfide, thereby forming detectably tagged protein substrate;
- detecting the detectablv tagged protein substrate, wherein the detectablv tagged protein substrate indicates that the protein substrate in the test sample was nitrosylated.
Dependencies are inferred from the claim text.