The goal of this Phase I STTR is to develop the gelbrane, a precast polyacrylamide gel combined with a transfermembrane, and a transfer apparatus. Western blotting, one of the most widely used protein assays in biomedicalresearch, enables detection of specific proteins and their post-translational modifications in blood, tissue, or celllysate samples via molecular weight-based separation. The technique has remained practically identical to whenit was first introduced in the late 1970s, and is often considered a gold standard for protein analysis. Followingseparation of lysate proteins by polyacrylamide gel electrophoresis, proteins are transferred onto a PVDF(polyvinylidene difluoride) or nitrocellulose membrane. A major error-prone step is construction of a "transfersandwich", where a membrane, blotter paper, and sponges are manually arranged around the gel followingelectrophoresis. Errors here often result in a completely failed experiment that is only discovered after ~2-3 days,causing sample loss, increased labor, lost time, and poor reproducibility. Current western blotting methodsmainly rely on immersed vertical electrophoresis, with sample wells traversing the entire gel thickness. In thisarrangement, a membrane already in close contact with a gel would non-specifically adsorb sample, renderingseparation practically useless. There is an unmet need for a product that eliminates the need for manual transfersandwich construction, while remaining affordable and familiar to investigators. We will develop two products:(i) a precast gelbrane cassette that contains a membrane already in perfect contact with a polyacrylamide geland is drop-in-ready for electrophoresis and transfer and (ii) a transfer apparatus for the gelbrane cassette thateliminates manual construction of a transfer sandwich. An enabling innovation is our prior horizontal tank-basedimmersed polyacrylamide gel electrophoresis (patent pending); only semi-dry horizontal or immersed verticaltank exist. This enables us to innovate precast gels by including a PVDF or nitrocellulose membrane during gelcasting, creating the gelbrane, which is "drop-in-ready" for our horizontal electrophoresis and transfer apparati.Phase I Hypothesis. Can a precast gelbrane cassette reliably undergo sample loading, electrophoresis, andtransfer? We hypothesize that by performing horizontal tank-based immersed electrophoresis, samples will notcome into contact with the membrane prior to transfer, enabling comparable results to gold-standard westernblotting with reduced probability for error. In Aim 1, we will establish robust electrophoresis with the gelbranecassette, focusing on 12 well gels and molecular weight ladder. In Aim 2, we will develop a robust gelbranetransfer apparatus, and use both molecular weight ladder and cell lysates. Success in each aim is defined byvariability (CV%) across analytes and technicians to be ~<10%. We expect to have a beta-testable product atthe end of Phase I. Our market is academic research labs and pre-clinical pharmaceutical 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. A manual intervention
at the "transfer" step can cause reproducibility issues and failed experiments only discovered days later. This
proposal develops a product that eliminates this manual intervention at transfer.
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