Accessible and Robust High-Throughput Western Blotting for Small Sample Sizes

项目摘要 本阶段I STTR的目标是Blotting Innovations，LLC建立可商业化的 mesowestern-我们最近开发的一种高通量、负担得起的蛋白质印迹技术。 蛋白质印迹法是一种分子量分辨分析蛋白质及其翻译后产物的技术。 今天的实践几乎与20世纪70年代末首次引入时相同。它仍然是 在生物医学研究中使用最广泛的蛋白质测定之一，也许是过去10年中使用最多的。 年主要原因是它成本低，通常是黄金标准，并且在大多数实验室中得到了很好的认可。然而，西方 印迹法难以按比例放大，通常每次运行限于~10个样品。基于毛细管电泳 已经开发了自动化装置中的分离，其增加了较小样品的生产量，但是 昂贵且对样品制备敏感。Microwestern使用压电移液器，最多可达 在标准占地面积中一次96个印迹;然而，压电设备强加了资本成本和技术成本。 难以遏制。我们建立了中尺度分析系统，分析了300多个具有相似足迹的样本， 与传统的Western印迹法相比，该方法具有经济实惠和易于使用的特点，并且样本量要求低约10倍。 我们的主要产品是一种预制的中分子凝胶，由低成本的移液机器人（opentrons）进行移液， 一种新颖但负担得起的槽，用于加载的预制凝胶的浸没式水平电泳。主要创新 是一种可定制的凝胶浇注装置，可生产数百个约1 μ L威尔斯孔的聚丙烯酰胺凝胶， 相关的方案，用于稳健的凝胶浇铸和电泳。另一个主要创新是浸入式水平 聚丙烯酰亚胺凝胶的槽电泳;仅半干水平（Microwestern）或浸入式垂直槽 （传统的）现在已经有了。第一阶段假设。预制介水凝胶是否能够牢固地加载 然后进行浸没式水平槽电泳？我们假设这可能是 通过设计在浇铸和运输期间保持凝胶的刚性插入物来实现，但也适合于（i） opentrons移液机器人和（ii）低成本，水平浸没电泳装置。我们有两个目标 通过测试这个假设来确定产品的可行性。在目标1中，我们将建立鲁棒的机器人 装载运输的预制中密度凝胶。我们专注于Opentrons微量移液机器人， 使用和非常实惠。在目标2中，我们将建立机器人加载的中分子筛的稳健电泳， 凝胶。每个目标的成功定义为分析物和技术人员之间的变异性（CV%）<10%。我们 希望在第一阶段结束时有一个可测试的产品。第二阶段将侧重于扩大到不同的 样本类型（例如免疫沉淀-Western）和跨抗体，以及稳健转移至 膜（另一个主要的变异性来源）。我们的市场是学术研究实验室和临床前 制药研发实验室

PROJECT SUMMARY The goal of this Phase I STTR is for Blotting Innovations, LLC to establish feasibility of a commercializable mesowestern—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-translational modifications that is practiced today almost identically to when it was first introduced in the late 1970s. It remains one of the most widely-used protein assays across biomedical research, perhaps the most used in the past 10 years. Major reasons are that it is low-cost, often a gold standard, and well-established in most labs. Yet, western blotting has been refractory to scale up, typically limited to ~10 samples per run. Capillary electrophoresis-based separation in automated apparati has been developed that increase throughput with smaller samples, but are expensive and can be sensitive to sample preparation. The microwestern uses piezoelectric pipetting for up to 96 blots at a time in a standard footprint; however, the piezoelectric apparatus imposes capital cost and technical difficultly 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) and a novel yet affordable tank for immersed horizontal electrophoresis of the loaded precast gel. A main innovation is a customizable gel casting device that produces polyacrylamide gels with hundreds of ~1 uL wells, and associated protocols for robust gel casting and electrophoresis. Another main innovation is immersed horizontal tank 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-loaded robotically, and then subjected to immersed horizontal tank electrophoresis? We hypothesize that this can be accomplished 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 Aims that will establish feasibility of the product by testing this hypothesis. In Aim 1, we will establish robust robotic loading of shipped, precast mesowestern gels. We focus on Opentrons micropipetting robots that are easy to use and very affordable. In Aim 2, we will establish robust electrophoresis of robotically-loaded mesowestern gels. 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. Phase II would focus on expanding to different sample types (e.g. Immunoprecipitation-western) and across antibodies, as well as on robust transfer to membrane (another main variability source). Our market is academic research labs and pre-clinical pharmaceutical R&D labs.