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

我实验室的研究探索了使动物能够适应和忍受极端环境的生化适应，包括缺氧、零度以下的温度和水分限制。我们使用三种主要方法来探索自然现象下的代谢和基因适应，包括耐冻性、冬眠性、退化性和厌氧性：酶学、免疫印迹和基因表达研究我实验室所有工作的关键准备仪器是高速冷冻离心机，用于制备组织提取物，用于酶分析、蛋白质纯化、亚细胞分离、Western blotting和mRNA提取。对酶调节的研究构成了实验室研究的中心核心，包括对中枢代谢途径的许多酶的功能适应性的研究，以及对传递外部信号并通过共价修饰调节和协调代谢酶活动的蛋白激酶和蛋白磷酸酶的分析。支持所有酶学的主要分析仪器是分光光度计。目前，我的实验室正在经历这些关键仪器的严重缺陷；我们已有27年历史的高速离心机已经退役，三台微板光谱仪是过时的型号，由于持续的机械和软件问题经常出现故障。本申请旨在通过购买新的地板式冷藏高速Beckman Avanti J-E离心机(外加两个不同容量的转子)、用于酶的常规微孔板分光光度分析的Thermo Multiskan光谱仪和具有吸光度、荧光和发光功能的多功能微孔板读取器Thermo Varioskan Flash Reader来解决核心“主力”实验室仪器的这些严重缺陷。后一种仪器将极大地增强和扩展我们在酶学方面的分析能力，使我们能够利用新的荧光和发光分析方法。

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.