Enhanced Staining and Imaging of Electrophoretically Separated Membrane Proteins Solubilized by SMA/DIBMA Polymers

Recent advances in membrane protein biochemistry have enabled the isolation of complexes in detergent-free, near-native states using synthetic amphipathic copolymers such as styrene-maleic acid (SMA) and diisobutylene maleic acid (DIBMA). However, these polymers often interfere with conventional protein detection methods, particularly in SDS-PAGE and Clear Native PAGE (CN-PAGE), hindering visualization and quantification. Here, we systematically evaluated 15 staining and detection methods-including Coomassie Brilliant Blue, silver, zinc, copper, Ponceau S, and SYPRO fluorescent dyes-on proteins solubilized by 13 different agents, including detergent n-dodecyl-β-d-maltoside (DDM), five SMA variants, and three DIBMA variants, from bovine heart mitochondria and cyanobacterial thylakoids. A photochemical, stain-free detection method using trichloroethanol (TCE) and UV activation proved to be optimal. This method covalently labels solvent-accessible tryptophan and tyrosine residues, generating robust fluorescence signals that are unaffected by polymer interference. TCE-modified proteins display dual emission peaks at ∼460 nm and a shoulder near 490 nm, likely corresponding to tyrosine and tryptophan adducts, respectively. The polymer-insensitive nature of TCE labeling allows sharp band resolution, particularly for low molecular weight proteins, and is compatible with high-throughput microplate analysis. This approach significantly enhances the qualitative and quantitative assessment of membrane proteins solubilized in polymer nanodiscs, enabling improved detection sensitivity, reduced background, and precise visualization of subunits. By facilitating accurate biochemical characterization of membrane proteins in their native-like lipid environments, this method provides a powerful tool for structural and functional proteomics across diverse biological systems.