Peptide Synthesis and Purification Core Facility

肽合成与纯化核心设施

基本信息

批准号：
7466200
负责人：
G M ANANTHARAMAIAH
金额：
$ 11.95万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7466200
关键词：
Alzheimer&apos s Disease Amino Acids Apolipoproteins A Atherosclerosis Biology Blood Vessels Core Facility Cost Savings Country Cultured Cells Diabetes Mellitus Disease Drug or chemical Tissue Distribution Fatty Acids Glass Goals High Density Lipoproteins High Pressure Liquid Chromatography Home environment Inflammatory Label Laboratories Length Liquid Chromatography Literature Methods Numbers Operative Surgical Procedures Paper Peptide Synthesis Peptides Phase Philadelphia Plant Resins Procedures Process Proteins Publications Publishing Radiolabeled Reaction Recombinant Proteins Research Research Design Research Personnel Role Site Staging Structure System Technology Time United States National Institutes of Health Universities Washington Wisconsin base day fast protein liquid chromatography improved in vivo instrumentation mimetics peptide analog pressure programs radiotracer silochrome size synthetic peptide

项目摘要

A. Administration. This facility will provide synthetic peptides for studies described mainly in projects 2, 3 and 4. This core is directed by Dr. Anantharamaiah, and Dr. Palgunachari will assist in the day-to-day operation of this facility. This core facility has been highly productive and has supported this program project extensively and therefore, an important component of this program project application. B. Instrumentation. The facility possesses three Protein Technology automatic peptide synthesizers and one Advanced ChemTech (ACT 90) peptide synthesizer. While the Protein Technology peptide synthesizers can provide small amounts of the peptides (in mg quantities) in a shorter duration due to the addition of ten times excess of incoming amino acids on to the amino acid-resin, the ACT can provide 10g quantities of peptides but the process is slow since one amino acid is added per day after the completion of reaction is ascertained at each stage of the synthesis. Since the starting resin used in ACT 90 is more, we avoid adding more than the required excess of amino acid derivatives at each stage in order to save costs on amino acid derivatives. For the purification of peptides the core facility possesses three Beckman analytical HPLC and two preparative HPLC units (Beckman and Varian). In addition to this, the core facility utilizes BioRad FPLC systems that are connected to Michel Muller Medium Pressure Liquid Chromatography (MPLC) glass columns. These columns are of various sizes with the smallest (analytical) containing 12g of the stationary phase (C-4 or C-18 silica gel) and the biggest column containing 800g of "home made" reverse phase stationary phase (C-4 and C-18 Reversed Phase silica gels). We routinely alkylate Silica Gel with various chain-lengths of fatty acids. At a time we produce 2 kg to 4kg of the stationary phase for home made columns. This provides a higher capacity to purify large quantities of synthetic peptides. These columns are also used to purify recombinant proteins. A typical procedure will be described in the Methods Section.

A、行政管理。该设施将为主要描述的研究提供合成肽项目 2、3 和 4。该核心由 Anantharamaiah 博士指导，Palgunachari 博士将协助该设施的日常运营。该核心设施生产力很高，并且广泛支持该计划项目，因此是该计划的重要组成部分项目申请。 B、仪器仪表。该设施拥有三台Protein Technology自动肽合成仪和一台 Advanced ChemTech (ACT 90) 肽合成仪。虽然蛋白质技术肽合成仪可以提供少量的肽（以毫克为单位）由于在氨基酸上添加了十倍过量的传入氨基酸，因此持续时间较短酸树脂，ACT可以提供10g量的肽，但过程很慢，因为一个氨基酸在合成的每个阶段确定反应完成后，每天添加酸。由于ACT 90中使用的起始树脂较多，我们避免添加超过所需的过量树脂各阶段的氨基酸衍生物，以节省氨基酸衍生物的成本。对于肽纯化核心设施拥有三台贝克曼分析型高效液相色谱仪和两台制备型 HPLC 装置（Beckman 和 Varian）。除此之外，核心设施还利用 BioRad 连接到 Michel Muller 中压液相色谱的 FPLC 系统 (MPLC) 玻璃柱。这些色谱柱有各种尺寸，最小的（分析）包含固定相（C-4或C-18硅胶）12g，最大柱含800g “自制”反相固定相（C-4 和 C-18 反相硅胶）。我们通常用不同链长的脂肪酸对硅胶进行烷基化。我们一次生产 2 公斤 4kg 自制柱固定相。这提供了更高的净化能力合成肽的数量。这些柱也用于纯化重组蛋白。一个典型的程序将在方法部分进行描述。