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Protein electrotransfer in conventional western blotting facilitates detection of size-separated proteins by diffusive immunoprobing, as analytes are transferred from a small-pore sizing gel to a blotting membrane for detection. This additional transfer step can, however, impair detection sensitivity through protein losses and confound protein localization. To overcome challenges associated with protein transfer, in-gel immunoassays immobilize target proteins to the hydrogel matrix for subsequent in-gel immunoprobing. Yet, detection sensitivity in diffusive immunoprobing of hydrogels is determined by the gel pore size relative to the probe size, and in-gel immunoprobing results in (i) reduced in-gel probe concentration compared to surrounding free-solution, and (ii) slow in-gel probe transfer compared to immunocomplex dissociation. Here, we demonstrate electrotransfer probing for effective and rapid immunoprobing of in-gel immunoassays. Critically, probe (rather than target protein) is electrotransferred from an inert, large-pore 'loading gel' to a small-pore protein sizing gel. Electric field is used as a tuneable parameter for electromigration velocity, providing electrotransfer probing with a fundamental advantage over diffusive probing. Using electrotransfer probing, we observe 6.5 ± 0.1× greater probe concentration loaded in-gel in ∼82× time reduction, and 2.7 ± 0.4× less probe concentration remaining in-gel after unloading in ∼180× time reduction (compared to diffusive probing). We then apply electrotransfer probing to detect OVA immobilized in-gel and achieve 4.1 ± 3.4× greater signal-to-noise ratio and 30× reduction in total immunoprobing duration compared to diffusive probing. We demonstrate electrotransfer probing as a substantially faster immunoprobing method for improved detection sensitivity of protein sizing in-gel immunoassays.

Rapid electrotransfer probing for improved detection sensitivity in in-gel immunoassays