Experimental Approach and Validation
Preliminary tests indicated significant endogenous protein glutathionylation in murine macrophage RAW 264.7 cells. A sample of these cells were fixed with paraformaldehyde, blocked, reduced, tagged, and FITC labeled. Pronounced fluorescence was observed by fluorescent microscopy in the cytoplasmic compartment, whereas the nucleus was nonfluorescent (Figure 2A).
Omission of the reducing step, which would prevent the release of glutathione residues and the generation of free sulfhydryl groups, resulted in absence of fluorescence (Figure 2B), supporting efficiency of the blocking step. Conversely, when blocking was omitted, intense fluorescence was obtained in both the cytoplasmic and nuclear compartments (Figure 2C).
Untreated RAW 264.7 cells were also evaluated using flow cytometry. One group of cells was fluorescently stained without the reducing step, which gives minimal FITC fluorescence.
By flow cytometry, these cells were found to consist of a major population of low fluorescence cells and a smaller population with higher fluorescence (Figure 3, black). The smaller population most likely reflects cells with high autofluorescence, as is often associated with phagosomes in macrophages. Fluorescent analysis of S-glutathionylation, using standard assay conditions, demonstrated a pronounced right shift of fluorescence intensity for both populations of cells (Figure 3, red).
Biotinylated proteins can be separated by SDS-PAGE, transferred to nitrocellulose, then probed by an avidin overlay technique. Proteins in lysates of RAW 264.7 cells, processed using the S-glutathionylation detection protocol with or without the PSSG reducing step, were separated, transferred, overlaid with avidin-HRP, and developed by enhanced chemiluminescence. Several S-glutathionylated proteins were detected in the sample that was analyzed using the complete protocol.