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

适用于小样本量的易于使用且稳定的高通量蛋白质印迹法

• 批准号: 10545990
• 负责人: Marc R. Birtwistle
• 金额: $ 28.88万
• 依托单位: BLOTTING INNOVATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2024-09-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10545990
• 关键词: Address; Antibodies; Biological Assay; Biology; Biomedical Research; Capillary Electrophoresis; Capital; Collaborations; Coomassie blue; Devices; Electrophoresis; Familiarity; Fluorescence; Gel; Goals; Gold; Hand; Immunoprecipitation; Intervention; Knowledge; Letters; Lice; Measurement; Membrane; Methods; Molecular Weight; Outcome; Pain; Pharmacologic Substance; Phase; Post-Translational Protein Processing; Preparation; Process; Protein Analysis; Proteins; Protocols documentation; Publications; Refractory; Research; Robot; Robotics; Running; Sample Size; Sampling; Shipping; Ships; Signal Transduction; Small Business Technology Transfer Research; Source; Specificity; Techniques; Technology; Testing; Time; Trust; Western Blotting; Width; base; commercial application; cost; design; innovation; interest; novel; operation; polyacrylamide gels; pre-clinical; research and development; scale up; success; technological innovation

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.

项目摘要 I阶段I STTR的目的是将创新，LLC建立可行性 Mesosestern-我们最近开发的一种高通量，负担得起的蛋白质印迹技术。 蛋白质印迹是对蛋白质及其翻译后分子量分析分析的一种技术 今天进行的修改几乎与1970年代后期首次引入的修改相同。它仍然存在 生物医学研究中最广泛使用的蛋白质测定法之一，也许是过去10个 年。主要原因是它是低成本的，通常是黄金标准，并且在大多数实验室中都建立了良好的成绩。但是，西方 印迹是难治性的，以扩大规模，通常限制为每次运行约10个样本。基于毛细管电泳 已经开发了自动化设备中的分离，可以用较小的样品增加吞吐量，但 昂贵，可能对样品制备敏感。 MicroFestern使用压电移液最多 标准足迹一次一次96个印迹；但是，压电设备可能不可能资本成本和技术 我们建立了Mesosestern，分析了300多个样本，具有相似的占地面积， 负担能力和易用性作为传统的蛋白质印迹，样本量要求降低约10倍。 我们的主要产品是一种预制的中层凝胶，可通过低成本移动机器人（Opentrons）和 一种新型但负担得起的储罐，用于浸入的载荷预制凝胶的水平电泳。主要创新 是一种可自定义的凝胶铸造装置，可生产具有数百个〜1 ul井的聚丙烯酰胺凝胶，并且 耐用凝胶铸造和电泳的相关方案。另一个主要创新是浸入水平 聚丙烯岩凝胶的罐电泳；仅半干水平（微瓶）或沉浸式垂直储罐 （传统）目前可用。第一阶段假设。可以预制中凝胶的凝胶。 机器人，然后受到浸泡的水平储罐电泳？我们假设这可能是 通过设计刚性插入物在铸造和运输过程中固定凝胶的刚性插件来完成，但也适合（i） Opentrons移动机器人和（ii）低成本的水平浸泡电泳设备。我们有两个目标 这将通过检验该假设来确定产品的可行性。在AIM 1中，我们将建立强大的机器人 装运，预制介质凝胶的负载。我们专注于易于使用的OpenRONS微膜片机器人 使用且价格合理。在AIM 2中，我们将建立强大的机器人载马的电泳 凝胶。每个目标的成功都由分析物和技术人员之间的可变性（CV％）定义为<10％。我们 期望在第一阶段结束时拥有β检验的产品 样本类型（例如免疫沉淀 - 西方）和跨抗体，以及强大的转移到 膜（另一个主要可变性来源）。我们的市场是学术研究实验室和临床前 药品研发实验室。