Optimization of thylakoid solubilization for PSII-LHCII supercomplex isolation from shade-adapted Fittonia albivenis

Isolation of photosystem II (PSII)-light-harvesting complex II (LHCII) supercomplexes is essential for elucidating their structure and function. Although established protocols are available for sun-adapted plants, efficient isolation of PSII-LHCII supercomplexes from shade-adapted species with distinct photosynthetic characteristics remains challenging. Here, we show that conventional PSII-LHCII isolation strategies, including direct solubilization with n-dodecyl-α-D-maltoside (α-DDM) and BBY (Berthold, Babcock, and Yokum) particle-based approaches, showing limited ability to preserve intact antenna-core associations in the shade-adapted plant Fittonia albivenis (F. albivenis), resulting in substantial photosystem I (PSI) contamination or reduced LHCII retention. By systematically evaluating detergent-based solubilization strategies, we identify a combination of α-DDM and digitonin as an effective detergent system for maintaining PSII-LHCII organization. The combined use of α-DDM and digitonin for direct thylakoid membrane solubilization, coupled with optimized sucrose density gradient conditions, enabled the successful isolation of PSII-LHCII supercomplexes with improved antenna retention and reduced PSI co-isolation. The optimized conditions are further successfully applied to two shade-adapted plant species, Epipremnum aureum (E. aureum) and Hygrophila difformis (H. difformis), supporting the applicability of this approach beyond F. albivenis. Together, this study provides a practical strategy for isolating photosynthetic supercomplexes from shade-adapted species examined here and highlighting the importance of species-specific optimization of thylakoid membrane-protein isolation conditions.