Biochemical adaptation: microplate readers and superspeed centrifuge for studies in enzymology and metabolic regulation

生化适应：酶标仪和超高速离心机用于酶学和代谢调节研究

• 批准号: 344871-2007
• 负责人: Storey, Kenneth
• 金额: $ 2.68万
• 依托单位: Carleton University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2006
• 资助国家: 加拿大
• 起止时间: 2006-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=326189
• 关键词: Biochemical; adaptation; microplate; readers; superspeed

Research in my lab explores the biochemical adaptations that allow animals to adapt to and endure environmental extremes including oxygen deprivation, subzero temperatures, and water limitation. We explore the metabolic and gene adaptations that underlie natural phenomena including freeze tolerance, hibernation, estivation and anaerobiosis using three main approaches: enzymology, immunoblotting, and gene expression studies The key preparative instrument that underlies all of the work in my lab is a high speed refrigerated centrifuge which is used to prepare tissue extracts for enzyme assay, protein purification, subcellular fractionation, Western blotting and mRNA extraction. Studies of enzyme regulation form the central core of the lab's research including studies of the functional adaptations of many enzymes of central metabolic pathways as well as analysis of the protein kinase and protein phosphatase enzymes that transduce external signals and, via covalent modifications, regulate and coordinate the activities of metabolic enzymes. The primary analytical instrument underlying all enzymology is the spectrophotometer. Currently, my lab is experiencing critical deficiencies in these key instruments; our 27 year old high speed centrifuge has been decommissioned and three microplate spectrophotometers are obsolete models that break down frequently due to ongoing mechanical and software problems. The present application seeks to solve these critical deficiencies in core "workhorse" lab instruments with the purchase of a new floor model, refrigerated, high speed Beckman Avanti J-E centrifuge (plus 2 rotors of differing capacities), a Thermo Multiskan Spectrum Photometer for routine microplate-based spectrophotometric assay of enzymes, and a Thermo Varioskan Flash Reader which is a multifunctional microplate reader with absorbance, fluorescence and luminescence capabilities. This latter instrument will greatly enhance and extend our analytical capabilities in enzymology by allowing us to take advantage of novel fluorescence and luminescence assay methods.

我实验室的研究探索了生物化学适应，使动物能够适应和忍受极端环境，包括缺氧、零下温度和水限制。我们使用三种主要方法探索自然现象（包括耐冻性、冬眠、夏眠和厌氧）背后的代谢和基因适应：酶学、免疫印迹和基因表达研究。我实验室所有工作的关键制备仪器是高速冷冻离心机，用于制备组织提取物，用于酶测定、蛋白质纯化、 亚细胞分级分离、蛋白质印迹和 mRNA 提取。酶调节研究构成了实验室研究的核心，包括对中央代谢途径中许多酶的功能适应的研究，以及对转导外部信号并通过共价修饰调节和协调代谢酶活性的蛋白激酶和蛋白磷酸酶的分析。所有酶学的主要分析仪器是分光光度计。目前，我的实验室在这些关键仪器上遇到了严重缺陷；我们已有 27 年历史的高速离心机已退役，三台微孔板分光光度计已过时，由于持续存在的机械和软件问题而经常出现故障。本申请旨在解决核心“主力”实验室仪器中的这些关键缺陷，购买了新的落地型号、冷冻高速 Beckman Avanti J-E 离心机（加上 2 个不同容量的转子）、用于常规基于微孔板的分光光度测定酶的 Thermo Multiskan 光谱光度计以及 Thermo Varioskan Flash Reader（ 具有吸光度、荧光和发光功能的多功能酶标仪。后一种仪器将使我们能够利用新型荧光和发光测定方法，从而极大地增强和扩展我们在酶学方面的分析能力。