The goal of this Phase I STTR is for Blotting Innovations, LLC to establish feasibility of a commercializablemesowestern-a high-throughput, affordable western blotting technique that we recently developed.Western blotting is a technique for molecular-weight-resolved analysis of proteins and their post-translationalmodifications that is practiced today almost identically to when it was first introduced in the late 1970s. It remainsone of the most widely-used protein assays across biomedical research, perhaps the most used in the past 10years. Major reasons are that it is low-cost, often a gold standard, and well-established in most labs. Yet, westernblotting has been refractory to scale up, typically limited to ~10 samples per run. Capillary electrophoresis-basedseparation in automated apparati has been developed that increase throughput with smaller samples, but areexpensive and can be sensitive to sample preparation. The microwestern uses piezoelectric pipetting for up to96 blots at a time in a standard footprint; however, the piezoelectric apparatus imposes capital cost and technicaldifficultly deterrents. We established the mesowestern that analyzes over 300 samples with a similar footprint,affordability, and ease-of-use as traditional western blots, and with ~10-fold lower sample size requirements.Our main products are a precast mesowestern gel that is loadable by a low-cost pipetting robot (opentrons) anda novel yet affordable tank for immersed horizontal electrophoresis of the loaded precast gel. A main innovationis a customizable gel casting device that produces polyacrylamide gels with hundreds of ~1 uL wells, andassociated protocols for robust gel casting and electrophoresis. Another main innovation is immersed horizontaltank electrophoresis for polyacrylimide gels; only semi-dry horizontal (microwestern) or immersed vertical tank(traditional) are currently available. Phase I Hypothesis. Can precast mesowestern gels be robustly-loadedrobotically, and then subjected to immersed horizontal tank electrophoresis? We hypothesize that this can beaccomplished by designing a rigid insert that holds the gel during casting and shipping but also fits into (i)opentrons pipetting robots and (ii) a low-cost, horizontal immersed electrophoresis apparatus. We have two Aimsthat will establish feasibility of the product by testing this hypothesis. In Aim 1, we will establish robust roboticloading of shipped, precast mesowestern gels. We focus on Opentrons micropipetting robots that are easy touse and very affordable. In Aim 2, we will establish robust electrophoresis of robotically-loaded mesowesterngels. Success in each aim is defined by variability (CV%) across analytes and technicians to be <10%. Weexpect to have a beta-testable product at the end of Phase I. Phase II would focus on expanding to differentsample types (e.g. Immunoprecipitation-western) and across antibodies, as well as on robust transfer tomembrane (another main variability source). Our market is academic research labs and pre-clinicalpharmaceutical R&D labs.
Public Health Relevance Statement: PROJECT NARRATIVE
Proteins and their post-translational modifications functionally control most of biology. Western blotting is a
reliable method for analyzing them but has remained largely unchanged for over 40 years, and is low throughput.
This proposal develops a feasible product for the mesowestern technology, which enables high-throughput
western blotting with minimal deviation from the already trusted protocols.
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