Abstract
Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NFκB, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.
Highlights
Inflammation is recognized as a driver of several chronic diseases including cancer and heart disease[1,2]
Ethics Statement For peripheral blood mononuclear cells (PBMCs), healthy individuals who agreed to participate in this study provided written informed consent
The devices were fabricated using standard soft-lithography techniques as previously described[12].First, the transparency mask was printed from a CAD file of the microfluidic device
Summary
Inflammation is recognized as a driver of several chronic diseases including cancer and heart disease[1,2]. Many regulatory steps are involved, protein modifications are one of the defining features of inflammatory responses[3,4,5]. Since its inception in 1979, protein immunoblotting has become the standard technique for profiling proteins and protein modifications in molecular biology and clinical diagnostics[6]. Traditional protein immunoblotting is a powerful technique, it has several limitations including its slow throughput, the requirement for relatively large sample and antibody amounts, and the inability to probe for multiple proteins simultaneously[7]. To overcome some of the limitations of traditional protein immunoblotting, several variations have been introduced including membrane stripping and the use of fluorescent secondary antibodies
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