Mal tiempo pero buen ambiente

Abstract

We introduce and characterize multiplexed native Western blotting in an automated and unified microfluidic format. While slab gel Western blotting is slow and laborious, conventional multiplexed blotting ("reblotting": probing one sample with multiple antibodies) requires even more resources. Here we detail three key advances that enable an automated and rapid microfluidic alternative to slab gel reblotting. First, we introduce both assay and microdevice designs that integrate protein blotting against multiple antibody blotting regions with native polyacrylamide gel electrophoresis. This microfluidic integration strategy overcomes nonspecific material losses inherent to harsh antibody stripping steps typically needed for conventional reblotting; said conditions can severely limit analyte quantitation. Second, to inform rational design of the multiplexed microfluidic device we develop an analytical model for analyte capture on the blotting regions. Comparison to empirical observations is reported, with capture efficiencies of >85%. Third, we introduce label free detection that makes simultaneous and quantitative multiplexed measurements possible without the need for prelabeling of sample. Assay linear dynamic range spans 8-800 nM with assay completion in 5 min. Owing to the speed, automation, enhanced quantitation capability, and the difficulty of conventional slab gel Western reblotting, microfluidic multiplexed native Western blotting should find use in systems biology, in particular in analyses of protein isoforms and multimeric protein complexes.