Next Generation Western for high-throughput and high-content protein analysis

DESCRIPTION provided by applicant The identification and quantification of specific proteins in complex samples are critical processes in discovering understanding and ultimately addressing the underlying nature of human health and disease The western blot is a fundamental method used for these processes due to the sensitivity and specificity enabled by combining protein sizing with immuno detection Despite being widely used the western blot has limits related to required sample size throughput information content quantification and repeatability which hinder many research efforts An example is the challenge in determining expression levels of multiple proteins in the very small samples associated with pancreatic islets and mouse biopsies used in research related to diabetes and obesity Next Generation Western NGW is an innovative approach to alleviate limitations of traditional western blotting using a novel method that automates and miniaturizes the process of electrophoresis followed by direct blotting onto a moving capture medium for subsequent immuno probing and detection To demonstrate feasibility of the NGW technology the specific aims are to enable separation resolution comparable to current western blot performance develop a sample injection loading scheme to optimize limit of detection for a wide range of protein sizes and demonstrate the power of the technology by analyzing the total and phosphorylated expression levels of five proteins in a single cell lysate using a sample size fold smaller than required by current technology LI COR Biosciences is well established in the western blot market with worldwide sales and support of instrumentation and reagents After demonstrating feasibility a product based on this technology will be developed in collaboration with the Kennedy lab to enable western blots with significantly less sample nanograms instead of micrograms reduced complication automated separation transfer and improved throughput and content lanes per current western blot lane PUBLIC HEALTH RELEVANCE The western blot is a fundamental technique used to identify and quantify specific proteins in complex samples however the technique has significant limitations that hinder progress in research efforts aimed at understanding and curing various human health issues and diseases This proposal focuses on development of a novel microfluidic direct blotting technique to automate and miniaturize western blotting This technology will enable throughput and content beyond the capabilities of traditional western blotting such as quantification of multiple proteins in small pancreatic islet samples from mice used in research related to diabetes and obesity