Affinity-tag-based microfluidic protein isolation enables high-resolution Cryo-EM from minimal starting material

Cryo-EM has become central to high-resolution structure determination, but conventional sample preparation consumes substantial quantities of purified protein and relies on multi-step workflows that can destabilize sensitive complexes. Microfluidic approaches can downscale and accelerate these workflows while retaining the particle numbers required for single-particle analysis. Here, we present a generalized microfluidic isolation strategy that captures proteins directly from cell lysates or in vitro translation (IVT) reactions via genetically encoded tags rather than target-specific binders. Both affinity-based (ALFA-nanobody) and covalent (SpyTag3/SpyCatcher3) capture are supported. Specificity derives from two independent steps: (i) tag-mediated capture and concentration of the target on magnetically trapped beads, and (ii) photoelution of the bound protein from the bead surface. This suppresses non-specific carryover, a dominant concern at microfluidic surface-to-volume ratios. Using this approach, we isolated E. coli ferritin A, {beta}-galactosidase, and P. aeruginosa VgrG1 from less than 50 {micro}L of cell lysate or IVT reaction, obtaining reconstructions between 1.9 [A] and 2.6 [A] resolution with B-factors competitive with optimized conventional workflows. Tag-based microfluidic isolation thus provides a broadly applicable route to cryo-EM sample preparation, reducing sample volumes more than 1000-fold, shortening preparation times 3- to 10-fold, and enabling high-throughput structural screening from IVT reactions.