Gelbrane: Combined Gel and Membrane for Robust Western Blotting

Gelbrane：结合凝胶和膜实现稳健的蛋白质印迹

• 批准号: 10759072
• 负责人: Marc R. Birtwistle
• 金额: $ 29.77万
• 依托单位: BLOTTING INNOVATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-08 至 2024-08-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10759072
• 关键词: Acrylamides; Actins; Adsorption; Antibodies; Biological Assay; Biology; Biomedical Research; Blood; Capital; Cells; Chemistry; Clinical; Collaborations; Data; Detection; Diagnostic; Drops; Dryness; Electrophoresis; Gel; Goals; Immunoprecipitation; Industrialization; Intervention; Legal patent; Letters; Manuals; Marketing; Membrane; Methods; Molecular Weight; Outcome; Paper; Performance; Pharmacologic Substance; Phase; Play; Polyacrylamide Gel Electrophoresis; Porifera; Post-Translational Protein Processing; Probability; Production; Protein Analysis; Proteins; Protocols documentation; Publications; Pyroxylin; Reproducibility; Research; Research Personnel; Rest; Route; Sampling; Signal Transduction; Small Business Technology Transfer Research; Speed; Techniques; Technology; Testing; Thick; Time; Tissues; Trust; Variant; Western Blotting; Width; Work; commercial application; cost; design; experimental study; improved; innovation; interest; migration; operation; polyacrylamide gels; polyvinylidene fluoride; pre-clinical; research and development; success; technological innovation

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.

项目摘要 第一阶段STTR的目标是开发gelbrane，一种预制聚丙烯酰胺凝胶， 膜和转移装置。蛋白质印迹法是生物医学中最广泛使用的蛋白质检测方法之一， 研究，能够检测血液，组织或细胞中的特定蛋白质及其翻译后修饰 裂解物样品通过基于分子量的分离。该技术实际上与 它于20世纪70年代末首次引入，通常被认为是蛋白质分析的黄金标准。以下 通过聚丙烯酰胺凝胶电泳分离裂解物蛋白，将蛋白转移到PVDF上， （聚偏二氟乙烯）或硝酸纤维素膜。一个主要的容易出错的步骤是构造一个“transfer 三明治”，其中膜、吸墨纸和海绵被手动地布置在凝胶周围， 电泳这里的错误往往导致完全失败的实验，只有在2-3天后才被发现， 导致样品损失、劳动增加、时间损失和再现性差。目前的蛋白质印迹方法 主要依靠浸入式垂直电泳，样品威尔斯孔穿过整个凝胶厚度。在这 在这种布置中，已经与凝胶紧密接触的膜将非特异性地吸附样品， 分离实际上是无用的。对于消除手动转移需要的产品存在未满足的需求 三明治结构，同时保持负担得起的和熟悉的调查。我们将开发两种产品： (i)一种预制的凝胶膜盒，其中包含已经与聚丙烯酰胺凝胶完美接触的膜 并且是用于电泳和转移的即插即用的;以及（ii）用于凝胶膜盒的转移装置，其 消除了转移夹层的手工构造。一项使能创新是我们先前的水平罐式 浸入式聚丙烯酰胺凝胶电泳（专利申请中）;仅半干水平或浸入式垂直 坦克存在。这使我们能够通过在凝胶过程中加入PVDF或硝酸纤维素膜来创新预制凝胶 铸造，创造gelbrane，这是“drop-in-ready”为我们的水平电泳和转移设备. 第一阶段假设。预制凝胶膜盒能否可靠地进行样品加载、电泳和 转移？我们假设，通过进行水平槽式浸入电泳，样品将不会 在转移之前与膜接触，从而能够获得与金标准Western 错误概率降低的印迹。在目标1中，我们将用凝胶膜建立稳健的电泳， 试剂盒，侧重于12孔凝胶和分子量梯度。在目标2中，我们将开发一个鲁棒的gelbrane 转移装置，并使用分子量梯和细胞裂解物。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%）约<10%。我们希望有一个beta测试的产品， 第一阶段结束。我们的市场是学术研究实验室和临床前制药研发实验室。