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bioRxiv · 10.64898/2026.08.27.747539

Interval-zone free-flow electrophoresis as a charge-specific dimension for native isolation of thylakoid membrane protein complexes

Eichacker, L. A.·Weber, G.
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Abstract

Native polyacrylamide gel electrophoresis is the standard first analytical step after detergent solubilization of thylakoid membranes. It separates photosystem supercomplexes by size and shape, but it forces a polydisperse mixture of protein-detergent particles through a gel of limited pore diameter. Interval-zone free-flow electrophoresis (iZE-FFE) performs that first electrophoretic step in free solution, without ampholytes or immobilines, and returns the complexes as a liquid fraction series ordered by net charge. We describe the operational cycle of iZE, show that bromophenol blue reports the charge density of {beta}-dodecylmaltoside micelles, and apply iZE to Arabidopsis thaliana thylakoid extracts from two sequential solubilization campaigns. After a two-step digitonin then digitonin/{beta}-dodecylmaltoside extraction (experiment 6874), SDS-PAGE of the iZE fractions is crowded: high-molecular mass protein-lipid assemblies of photosystem I and photosystem II release the same subunits into many wells. Native-PAGE of the same fractions resolves ATP-synthase from PSI, a free LHCII pool, anodic PSII, and cathodic PSI, including PSI with and without LHCII. A three-step extraction with a single pH working window (experiment 7154) confirms this charge order. Lowering residual detergent in consecutive extracts still produces partly solubilized high-mass assemblies, whereas a final {beta}-dodecylmaltoside step releases the C2S2M2/C2S2M/C2S2 PSII series and a single PSI band. iZE is therefore a charge-first, orthogonal dimension for native thylakoid biochemistry. Native-PAGE, not SDS-PAGE, is the proper second dimension while solubilization remains incomplete.

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Eichacker, L. A., Weber, G.. 2026-08-28. Interval-zone free-flow electrophoresis as a charge-specific dimension for native isolation of thylakoid membrane protein complexes. https://doi.org/10.64898/2026.08.27.747539

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