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 该阶段I STTR的目的是开发凝胶，这是一种预制的聚丙烯酰胺凝胶，结合了转移 膜和转移设备。蛋白质印迹，是生物医学中使用最广泛的蛋白质测定法之一 研究，可以检测特定蛋白质及其在血液，组织或细胞中的翻译后修饰 裂解物样品通过基于分子量的分离。该技术与何时相同 它是在1970年代后期首次引入的，通常被认为是蛋白质分析的黄金标准。下列的 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 （聚偏二氟化物）或硝酸纤维素膜。容易出错的主要步骤是构建“转移 三明治”，在凝胶周围手动排列膜，斑点纸和海绵 电泳。这里的错误通常会导致完全失败的实验，该实验仅在约2-3天后才发现， 导致样本损失，劳动力增加，时间损失和可重复性差。当前的蛋白质印迹方法 主要依靠浸入式垂直电泳，样品井横穿整个凝胶厚度。在这个 布置，已经与凝胶紧密接触的膜将非特异性吸附样品，呈现 分离实际上有用。对于消除了手动转移需求的产品的需求未满足 三明治建设，同时保持负担得起和熟悉调查员。我们将开发两个产品： （i）一个预制的胶质盒，其中包含已经与聚丙烯酰胺凝胶完全接触的膜 并且已经准备好用于电泳和转移以及（ii）胶质盒的转移设备 消除转移三明治的手动构造。启用创新是我们先前基于水平坦克的 浸入聚丙烯酰胺凝胶电泳（申请专利）；仅半干水平或沉浸式垂直 坦克存在。这使我们能够通过在凝胶期间包括PVDF或硝酸纤维素膜来使预制凝胶无效 铸造，创建胶质，这是我们水平电泳和转移设备的“准备就绪”。 第一阶段假设。可以可靠地进行样品加载，电泳和 转移？我们假设通过执行水平储罐的沉浸电泳，样品将不会 转移前与膜接触，从 误差概率降低。在AIM 1中，我们将用胶质建立强大的电泳 盒式磁带，重点放在12孔凝胶和分子量层压板上。在AIM 2中，我们将开发出强大的凝胶 转移设备，并同时使用分子量层压板和细胞裂解物。每个目标的成功都由 分析物和技术人员之间的可变性（CV％）约为<10％。我们希望在 第1阶段的结束。我们的市场是学术研究实验室和临床前药物研发实验室。