PROJECT SUMMARY The goal of this Phase I STTR is to develop the gelbrane, a precast polyacrylamide gel combined with a transfer membrane, and a transfer apparatus. Western blotting, one of the most widely used protein assays in biomedical research, enables detection of specific proteins and their post-translational modifications in blood, tissue, or cell lysate samples via molecular weight-based separation. The technique has remained practically identical to when it was first introduced in the late 1970s, and is often considered a gold standard for protein analysis. Following separation 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 “transfer sandwich”, where a membrane, blotter paper, and sponges are manually arranged around the gel following electrophoresis. 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 methods mainly rely on immersed vertical electrophoresis, with sample wells traversing the entire gel thickness. In this arrangement, a membrane already in close contact with a gel would non-specifically adsorb sample, rendering separation practically useless. There is an unmet need for a product that eliminates the need for manual transfer sandwich 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 gel and is drop-in-ready for electrophoresis and transfer and (ii) a transfer apparatus for the gelbrane cassette that eliminates manual construction of a transfer sandwich. An enabling innovation is our prior horizontal tank-based immersed polyacrylamide gel electrophoresis (patent pending); only semi-dry horizontal or immersed vertical tank exist. This enables us to innovate precast gels by including a PVDF or nitrocellulose membrane during gel casting, 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, and transfer? We hypothesize that by performing horizontal tank-based immersed electrophoresis, samples will not come into contact with the membrane prior to transfer, enabling comparable results to gold-standard western blotting with reduced probability for error. In Aim 1, we will establish robust electrophoresis with the gelbrane cassette, focusing on 12 well gels and molecular weight ladder. In Aim 2, we will develop a robust gelbrane transfer apparatus, and use both molecular weight ladder and cell lysates. Success in each aim is defined by variability (CV%) across analytes and technicians to be ~<10%. We expect to have a beta-testable product at the end of Phase I. Our market is academic research labs and pre-clinical pharmaceutical R&D labs.

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.