Gelbrane: Combined Gel and Membrane for Robust Western Blotting

项目总结 第一阶段的目标是开发凝胶膜，一种预制的聚丙烯酰胺凝胶，结合转移 以及一种转移装置。蛋白质印迹--生物医学中应用最广泛的蛋白质分析方法之一 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 裂解样品通过基于分子量的分离。这项技术实际上保持了与 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。跟随 用聚丙烯酰胺凝胶电泳法分离裂解蛋白，将蛋白质转移到PVDF上 (聚偏二氟乙烯)或硝化纤维素膜。一个容易出错的主要步骤是构造一个 三明治“，将薄膜、吸墨纸和海绵手动放置在凝胶周围 电泳法。这里的错误通常会导致实验完全失败，只有在大约2-3天后才能发现， 造成样品丢失、人工增加、时间浪费、重复性差。当前的西方印迹方法 主要依靠浸泡垂直电泳法，用样品井遍历整个凝胶厚度。在这 安排，已经与凝胶紧密接触的膜将非特异性地吸附样品，呈现 分离几乎毫无用处。存在对消除手动转移需要的产品的未满足的需求 三明治结构，同时保持负担得起和调查人员熟悉。我们将开发两个产品： (I)预制胶膜盒，内含已与聚丙烯酰胺凝胶完全接触的膜 以及(Ii)一种用于胶膜盒的传送装置，其 消除了手动构建转移夹层。一项令人信服的创新是我们以前基于水平坦克的 浸泡聚丙烯酰胺凝胶电泳法(专利申请中)；仅适用于半干水平或垂直浸泡 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，创造凝胶薄膜，这是“下降的准备”，为我们的水平电泳和转移设备。 第一阶段假设。预制的胶膜盒能否可靠地进行样品加载、电泳和 调职？我们假设，通过进行基于水平槽的浸泡电泳，样品不会 在转移前与膜接触，使结果与金标Western相当 减少了错误概率的印迹。在目标1中，我们将用凝胶膜建立健壮的电泳法 盒式磁带，重点是12井凝胶和分子量阶梯。在目标2中，我们将开发一种坚固的凝胶薄膜 转移装置，并使用分子量梯形图和细胞裂解物。每个目标的成功是由以下因素定义的 分析人员和技术人员之间的可变性(CV%)为10%。我们希望在以下位置推出可测试的产品 一期结束，我们的市场是学术研究实验室和临床前药物研发实验室。

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.