Immunofluorescent detection of S-nitrosoproteins in cell culture

The role of S-nitrosylation in cellular signaling has been clearly demonstrated. There a number of mechanisms whereby this post-translational modification can occur and the number of protein targets continue to expand. The need to be able to monitor when this important signaling process occurs within cells is increasingly important. Previously we have identified immunohistochemistry approaches effective for monitoring S-nitrosylation within fixed tissue. Within this paper we show how these techniques can be adapted to use in a cell culture system using immunofluorescence. We have used this protocol to detect S-nitrosoprotein formation within LPS stimulated microglial cells using both transformed and primary cultured cells.