Mourdoukoutas Andoni P, Grist Samantha M, Herr Amy E
The UC Berkeley/UCSF Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, California 94720, USA.
Anal Methods. 2020 Oct 8;12(38):4638-4648. doi: 10.1039/d0ay01203c.
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.
在传统的蛋白质免疫印迹法中，蛋白质电转印有助于通过扩散免疫检测来检测经大小分离的蛋白质，因为分析物会从小孔筛分凝胶转移至印迹膜上进行检测。然而，这一额外的转移步骤可能会因蛋白质损失而降低检测灵敏度，并使蛋白质定位变得复杂。为了克服与蛋白质转移相关的挑战，凝胶内免疫测定法将目标蛋白质固定在水凝胶基质上，以便随后进行凝胶内免疫检测。然而，水凝胶扩散免疫检测的灵敏度取决于凝胶孔径与探针大小的相对关系，并且凝胶内免疫检测会导致：（i）与周围自由溶液相比，凝胶内探针浓度降低；（ii）与免疫复合物解离相比，凝胶内探针转移缓慢。在此，我们展示了用于凝胶内免疫测定法有效且快速免疫检测的电转印检测法。关键在于，探针（而非目标蛋白质）从惰性大孔“上样凝胶”电转移至小孔蛋白质筛分凝胶。电场用作电迁移速度的可调参数，这使得电转印检测法相对于扩散检测法具有根本优势。使用电转印检测法，我们观察到在凝胶中加载的探针浓度提高了6.5±0.1倍，时间减少了约82倍，卸载后凝胶中剩余的探针浓度减少了2.7±0.4倍，时间减少了约180倍（与扩散检测法相比）。然后，我们应用电转印检测法来检测固定在凝胶内的卵清蛋白（OVA），与扩散检测法相比，实现了信噪比提高4.1±3.4倍，总免疫检测持续时间缩短30倍。我们证明电转印检测法是一种显著更快的免疫检测方法，可提高凝胶内蛋白质筛分免疫测定法的检测灵敏度。
