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
基本信息
- 批准号:10759072
- 负责人:
- 金额:$ 29.77万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AcrylamidesActinsAdsorptionAntibodiesBiological AssayBiologyBiomedical ResearchBloodCapitalCellsChemistryClinicalCollaborationsDataDetectionDiagnosticDropsDrynessElectrophoresisGelGoalsImmunoprecipitationIndustrializationInterventionLegal patentLettersManualsMarketingMembraneMethodsMolecular WeightOutcomePaperPerformancePharmacologic SubstancePhasePlayPolyacrylamide Gel ElectrophoresisPoriferaPost-Translational Protein ProcessingProbabilityProductionProtein AnalysisProteinsProtocols documentationPublicationsPyroxylinReproducibilityResearchResearch PersonnelRestRouteSamplingSignal TransductionSmall Business Technology Transfer ResearchSpeedTechniquesTechnologyTestingThickTimeTissuesTrustVariantWestern BlottingWidthWorkcommercial applicationcostdesignexperimental studyimprovedinnovationinterestmigrationoperationpolyacrylamide gelspolyvinylidene fluoridepre-clinicalresearch and developmentsuccesstechnological innovation
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Marc R. Birtwistle其他文献
Purifying circular RNA by ultrafiltration
通过超滤纯化环状RNA
- DOI:
10.1016/j.seppur.2025.132809 - 发表时间:
2025-08-27 - 期刊:
- 影响因子:9.000
- 作者:
Karen Guillen-Cuevas;Xiaoming Lu;Marc R. Birtwistle;Scott M. Husson - 通讯作者:
Scott M. Husson
Theory for High-Throughput Genetic Interaction Screening
高通量遗传相互作用筛选理论
- DOI:
10.1021/acssynbio.2c00627 - 发表时间:
2023-08-18 - 期刊:
- 影响因子:3.900
- 作者:
Madeline E. McCarthy;William B. Dodd;Xiaoming Lu;Daniel J. Pritko;Nishi D. Patel;Charlotte V. Haskell;Hugo Sanabria;Mark A. Blenner;Marc R. Birtwistle - 通讯作者:
Marc R. Birtwistle
Network analyses of brain tumor multiomic data reveal pharmacological opportunities to alter cell state transitions
对脑瘤多组学数据的网络分析揭示了改变细胞状态转变的药理学机会
- DOI:
10.1038/s41540-025-00493-2 - 发表时间:
2025-02-01 - 期刊:
- 影响因子:3.500
- 作者:
Brandon Bumbaca;Jonah R. Huggins;Marc R. Birtwistle;James M. Gallo - 通讯作者:
James M. Gallo
Marc R. Birtwistle的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('Marc R. Birtwistle', 18)}}的其他基金
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 - 财政年份:2022
- 资助金额:
$ 29.77万 - 项目类别:
Mechanistic Pharmacodynamic Modeling for Drug Combination Responses
药物组合反应的机制药效学建模
- 批准号:
10398952 - 财政年份:2021
- 资助金额:
$ 29.77万 - 项目类别:
Mechanistic Pharmacodynamic Modeling for Drug Combination Responses
药物组合反应的机制药效学建模
- 批准号:
10580895 - 财政年份:2021
- 资助金额:
$ 29.77万 - 项目类别:
Mechanistic Pharmacodynamic Modeling for Drug Combination Responses
药物组合反应的机制药效学建模
- 批准号:
10592423 - 财政年份:2021
- 资助金额:
$ 29.77万 - 项目类别:
Mechanistic Pharmacodynamic Modeling for Drug Combination Responses
药物组合反应的机制药效学建模
- 批准号:
10206849 - 财政年份:2021
- 资助金额:
$ 29.77万 - 项目类别:
Administrative Supplement to Support Summer Undergraduate Research for the Parent MIRA Award R35 GM141891 “Mechanistic Pharmacodynamic Modeling for Drug Combinations"
支持家长 MIRA 奖 R35 GM141891 暑期本科生研究的行政补充 — 药物组合的机械药效学建模”
- 批准号:
10809119 - 财政年份:2021
- 资助金额:
$ 29.77万 - 项目类别:
Multiplexed, Quantitative Fluorescence Imaging in Tumor Sections
肿瘤切片的多重定量荧光成像
- 批准号:
9566479 - 财政年份:2015
- 资助金额:
$ 29.77万 - 项目类别:
Multiplexed, Quantitative Fluorescence Imaging in Tumor Sections
肿瘤切片的多重定量荧光成像
- 批准号:
9329290 - 财政年份:2015
- 资助金额:
$ 29.77万 - 项目类别:
Multiplexed, Quantitative Fluorescence Imaging in Tumor Sections
肿瘤切片的多重定量荧光成像
- 批准号:
8928922 - 财政年份:2015
- 资助金额:
$ 29.77万 - 项目类别:
Drug Combination Signatures for Prediction and Mitigation of Toxicity
用于预测和减轻毒性的药物组合特征
- 批准号:
8787833 - 财政年份:2014
- 资助金额:
$ 29.77万 - 项目类别:
相似海外基金
A novel motility system driven by two classes of bacterial actins MreB
由两类细菌肌动蛋白 MreB 驱动的新型运动系统
- 批准号:
22KJ2613 - 财政年份:2023
- 资助金额:
$ 29.77万 - 项目类别:
Grant-in-Aid for JSPS Fellows
The structural basis of plasmid segregation by bacterial actins
细菌肌动蛋白分离质粒的结构基础
- 批准号:
342887 - 财政年份:2016
- 资助金额:
$ 29.77万 - 项目类别:
Operating Grants
The structural basis for plasmid segregation by bacterial actins
细菌肌动蛋白分离质粒的结构基础
- 批准号:
278338 - 财政年份:2013
- 资助金额:
$ 29.77万 - 项目类别:
Operating Grants
Cytoplasmic Actins in Maintenance of Muscle Mitochondria
细胞质肌动蛋白在维持肌肉线粒体中的作用
- 批准号:
8505938 - 财政年份:2012
- 资助金额:
$ 29.77万 - 项目类别:
Differential Expression of the Diverse Plant Actins
多种植物肌动蛋白的差异表达
- 批准号:
7931495 - 财政年份:2009
- 资助金额:
$ 29.77万 - 项目类别:
Studies on how actins and microtubules are coordinated and its relevancy.
研究肌动蛋白和微管如何协调及其相关性。
- 批准号:
19390048 - 财政年份:2007
- 资助金额:
$ 29.77万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Interaction of myosin with monomeric actins
肌球蛋白与单体肌动蛋白的相互作用
- 批准号:
5311554 - 财政年份:2001
- 资助金额:
$ 29.77万 - 项目类别:
Priority Programmes
STRUCTURE/INTERACTIONS OF ACTINS AND ACTIN-BINDING PROTEIN
肌动蛋白和肌动蛋白结合蛋白的结构/相互作用
- 批准号:
6316669 - 财政年份:2000
- 资助金额:
$ 29.77万 - 项目类别: