The reverse staining, with imidazole-SDS-zinc, of PEG-linked proteins separated by SDS-PAGE was studied. Using model conjugates (interferon-alpha 2b (IFN-alpha 2b) reacted with either a branched-chain (40000) PEG (PEG(2,40)) or a linear monomethoxy PEG polymer (M, of 12 000) and chromatographically purified mono P E G(2,40)-IFN-alpha 2b), conventional small-format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate-generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse-stained PEGylated IFN-alpha 2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (12). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono- or bi-PEG(2,40) IFN-alpha 2b per lane on gradient (4-17%) gels). Also, this stain allowed the visualization of Coomassie blue-undetected PEG-IFN bands, and could be restained with I-2. PEGylated species of lysozyme, a low-molecular-weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4-6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse-stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography-based positional isomer separation profile and enzyme-linked immunosorbent response of PEG-IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small-scale preparation of PEGylated proteins.