Sensitive detection of protein-protein and nucleic acid-protein interactions using a digital imaging system

使用数字成像系统灵敏检测蛋白质-蛋白质和核酸-蛋白质相互作用

• 批准号: RTI-2022-00427
• 负责人: Pelka, Peter
• 金额: $ 4.57万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743066
• 关键词: Sensitive; detection; protein; nucleic; acid

Molecular studies of protein-protein interactions have shed invaluable light on a multitude of cellular processes and pathways, ranging from the molecular regulation of intracellular signalling to large-scale understanding of organismal development and life processes. Likewise, studies of how proteins interact with nucleic acids have re-written textbooks and revolutionized our understanding of nucleic acids, both at the level of the nucleic acid itself and at the higher levels of chromatin structure and organization. Mechanistic insights gained from such studies have illuminated our understanding of countless processes and how they regulate life itself. Importantly, work carried out by the Pelka and Kumar groups have used such molecular biology expertise to probe pathways regulating host-pathogen interactions and molecular pathways conferring antibiotic resistance. Our NSERC-funded research focuses on the understanding of how viral proteins reprogram cellular networks during infection and how bacteria disarm antibiotics. This has led to a clearer understanding of not only the mechanisms by which these parasites overtake a host cell, but also have shed important insight into a variety of cellular regulatory networks and the mechanisms by which these networks are controlled, both at the level of the host cell and also within the parasite itself. We depend on an aging Konica SRX 101-A developer with X-ray film for our protein, nucleic acid, and protein-nucleic acid detection needs. It is the key to much of our NSERC-funded research success. Without the ability to detect proteins or nucleic acids we cannot complete the majority of our NSERC Discovery Grant goals. Unfortunately, the system is so old that Konica no longer supports it or manufactures spare parts, as such repairs and service lifetime is very limited while replacement parts are available. In addition, supplies necessary to operate the film developer have become expensive and more difficult to acquire; for example, the developer and fixer chemicals are no longer available in Manitoba and we import them from Ontario at greater cost. Likewise, the costs of film have risen dramatically over the last five years and with the number of western blots we routinely perform, the expense of carrying out these experiments have soared. Combined with frequent down-times due to lack of chemicals, film, or equipment breakdown, this has greatly compromised our ability to perform certain types of assays that rely on sensitive detection of proteins or nucleic acids while harming training of HQP. Ultimately, unrecoverable failure of this system is imminent. Thus, we urgently seek to replace our obsolete film-based Konica SRX 101-A with a new Azure 600 digital imaging system to continue our research and HQP training without fear of irrecoverable equipment failure. Crucially, there is no alternative for us to use and the looming failure of our developer will deprive us of the ability to image blots.

蛋白质-蛋白质相互作用的分子研究揭示了许多细胞过程和途径的宝贵信息，从细胞内信号的分子调控到对生物发育和生命过程的大规模理解。同样，对蛋白质如何与核酸相互作用的研究重写了教科书，并彻底改变了我们对核酸的理解，无论是在核酸本身的水平上，还是在染色质结构和组织的更高水平上。从这类研究中获得的机械论见解启发了我们对无数过程的理解，以及它们是如何调节生命本身的。重要的是，Pelka和Kumar小组开展的工作利用了这种分子生物学专业知识来探索调节宿主-病原体相互作用的途径和导致抗生素耐药性的分子途径。我们由NSERC资助的研究专注于了解病毒蛋白在感染过程中如何重新编程细胞网络，以及细菌如何解除抗生素的作用。这不仅使人们对这些寄生虫侵占宿主细胞的机制有了更清晰的了解，而且对各种细胞调控网络以及这些网络在宿主细胞水平和寄生虫自身内部的控制机制也有了重要的见解。我们依靠陈旧的柯尼卡SRX 101-A显影剂和X射线胶片来满足我们的蛋白质、核酸和蛋白质-核酸检测需求。这是我们NSERC资助的大部分研究成功的关键。如果没有检测蛋白质或核酸的能力，我们就不能完成NSERC发现拨款的大部分目标。不幸的是，该系统太旧了，柯尼卡不再支持它或制造备件，因为在有更换部件的情况下，这种维修和使用寿命非常有限。此外，操作胶片显影剂所需的用品已经变得昂贵，更难获得；例如，马尼托巴省不再有显影剂和定影剂化学品，我们以更高的成本从安大略省进口它们。同样，胶片成本在过去五年里大幅上升，随着我们经常进行的西方印迹的数量增加，进行这些实验的费用飙升。再加上由于缺乏化学品、胶片或设备故障而导致的频繁停机，这极大地影响了我们执行某些类型的分析的能力，这些分析依赖于对蛋白质或核酸的灵敏检测，同时损害了HQP的培训。最终，这个系统的不可挽回的故障迫在眉睫。因此，我们迫切希望用新的Azure 600数字成像系统替换我们过时的胶片柯尼卡SRX 101-A，以便继续我们的研究和HQP培训，而不必担心无法恢复的设备故障。至关重要的是，我们别无选择，我们的开发人员即将失败，这将剥夺我们成像斑点的能力。