PROTACs have rapidly matured into a leading modality as proximity therapeutics, leveraging the ubiquitin–proteasome system to eliminate proteins by enforcing proximity between an E3 ligase and a protein of interest. Mechanistically, their activity is governed not only by binary affinities, but by the formation, stability, and productive geometry of the ternary complex.
In vitro, however, activity and tractability often hinge on a deceptively simple step – formation of a productive E3-PROTAC-target ternary complex. Even when binary binding is detectable, ternary assembly can be limited by cooperativity, kinetic barriers, conformational heterogeneity, and competition from non‑productive binary species. As a result, apparently “small” experimental choices (ratios, order of addition, incubation time, buffer composition, and purification strategy) can dominate whether you recover a discrete, well‑behaved complex.
For one of our incubation set-ups we found that assembling the reaction with target protein and PROTAC in defined molar excess (4x in the example shown) increased the population of the ternary species captured by SEC. Equally important was column selection: matching the SEC matrix to the hydrodynamic size of the complex improved resolution and enabled efficient removal of excess unbound protein. This substantially improved recovery and sample homogeneity.
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