Chemiluminescent Labeling and Detection in Gels

凝胶中的化学发光标记和检测

• 批准号: 6444421
• 负责人: Hashem Akhavan-Tafti
• 金额: $ 10万
• 依托单位: LUMIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2002-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6444421
• 关键词: affinity labeling; chemical conjugate; chemical kinetics; chemical synthesis; gel electrophoresis; luminescence; nucleic acid quantitation /detection; technology /technique development

The objective of Phase I is to prepare a new family of chemiluminescent label compounds which can be conjugated to proteins and nucleic acids, subjected to electrophoresis and triggered to generate chemiluminescence in the gel. Successful development of the labeling compounds will permit the development of simple methods for direct detection in gels by eliminating membrane transfer and immunological binding protocols used in many techniques. The patented chemiluminescent technology involves reaction of the compounds with inexpensive chemical reagents. A prototypical compound produces visible chemiluminescence within an electrophoresis gel when conjugated to a protein. Additional chemiluminescent compounds emitting at distinct wavelengths spanning the visible spectrum will be synthesized and tested. Synthetic methods for conjugation will be established. Linkers for coupling to amine and thiol groups and a phosphoramidite linker are targeted. The chemiluminescence intensity, kinetics and spectrum of the chemiluminescent compounds will be evaluated. A key feature of the chemiluminescent reaction is that light is emitted as a 1-2 second burst when triggered in solution so that light intensity will be as high as possible when triggering is conducted within a gel. Detection sensitivity in solution and gel matrices will then be assessed using labeled analytes in model assays. PROPOSED COMMERCIAL APPLICATION: The proposed research seeks to develop a family of chemiluminescent direct labels which will enable the rapid and sensitive separation and detection of labeled protein and nucleic acid analytes within electrophoresis gels. The new method would greatly simplify analyses which now rely on membrane transfer or immunological binding protocols. Envisioned uses include preparing labeled polypeptide and nucleic acid size markers, 2D protein analysis, electrophoretic mobility shift analysis of transcriptional factors, nucleic acid hybridization assays and analysis of gene expression levels.

第一阶段的目标是制备一类新的化学发光标记化合物，这些化合物可以结合到蛋白质和核酸上，经过电泳并在凝胶中触发产生化学发光。标记化合物的成功开发将允许通过消除许多技术中使用的膜转移和免疫结合协议，开发直接检测凝胶的简单方法。该专利化学发光技术涉及化合物与廉价化学试剂的反应。一种典型的化合物与蛋白质结合后，在电泳凝胶中产生可见的化学发光。额外的化学发光化合物在不同的波长发射跨越可见光谱将被合成和测试。建立偶联的合成方法。偶联到胺和巯基的连接剂和磷酰胺连接剂是目标。对化学发光化合物的化学发光强度、动力学和光谱进行了评价。化学发光反应的一个关键特征是，当在溶液中触发时，光以1-2秒的爆发形式发射，因此当在凝胶中进行触发时，光强度将尽可能高。然后在模型分析中使用标记分析物评估溶液和凝胶基质中的检测灵敏度。拟议的商业应用：拟议的研究旨在开发一系列化学发光直接标签，这将使电泳凝胶中标记的蛋白质和核酸分析物能够快速、灵敏地分离和检测。新方法将大大简化目前依赖于膜转移或免疫结合协议的分析。设想的用途包括制备标记多肽和核酸大小标记，二维蛋白质分析，转录因子的电泳迁移率转移分析，核酸杂交分析和基因表达水平分析。