CORE--BIOCHEMISTRY

核心--生物化学

• 批准号: 5213430
• 负责人: A G SCICLI
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5213430
• 关键词: affinity chromatography; analytical chemistry; antibody; antireceptor antibody; autoradiography; biomedical facility; clone cells; dogs; gel electrophoresis; high performance liquid chromatography; hybridomas; kallikreins; laboratory mouse; laboratory rabbit; laboratory rat; monoclonal antibody; northern blottings; polymerase chain reaction; protein purification; reversed phase chromatography; southern blotting; tissue /cell culture; western blottings

The purpose of the Biochemistry Core laboratory is to centralize tasks such as protein purification, production and characterization of antibodies, preparation of Fab fragments of antibodies, and analysis requiring high performance liquid chromatography (HPLC). In addition, the core will set up routine analytical techniques. We will: I): a) maintain stable supplies of dog kininogens to be used in kininogenase assays and maintain stable supplies of rat glandular kallikrein, SEV and related proteins. b) produce polyclonal antibodies against peptides present in external domains of the kinin receptor and prepare and purify antibodies against rat glandular kallikrein and submandibular enzymatic vasoconstrictor. Antibodies will be used to perform or develop RIAs and for immunocytochemical localization studies. II): maintain hybridomas and produce and purify monoclonal antibodies and Fab fragments to proteins and peptides as required by the different project investigators. We will maintain supplies of monoclonal antibodies against kinins, ANF and rat glandular kallikrein, purify immunoglobulins and produce the corresponding Fab fragments. Antibodies will be produced in large quantities using the Bioreactor technique. III) employ reverse-phase and gel-exclusion HPLC techniques to identify and analyze peptides. IV): perform analytical and quantitative molecular biology techniques as required by participant investigators. Techniques requiring polyacrylamide gel electrophoresis, blotting and autoradiography will also be performed by the Core. V): a) maintain established cell lines in culture. Cell lines, such as M1 and bovine pulmonary artery endothelial cells, will be maintained as required by participant investigators. VI): perform analytical techniques such as measurement of enzymatic activities, protein, electrolytes, inulin and p- amino hippurate. Close communication among the project investigators and Biochemistry Core personnel will permit efficient planning and allow better coordination of the research efforts. Methodological variability will be minimized, and integration of biological data will be enhanced. In this way we will increase efficiency and reduce costs.

生物化学核心实验室的目的是集中以下任务 随着蛋白质的提纯、抗体的产生和鉴定， 抗体Fab片段的制备及高要求分析 高效液相色谱分析。此外，核心将设置 常规的分析技术。我们将：一)：一)保持稳定 用于激肽原酶分析和维护的犬激肽原供应 大鼠腺激肽释放酶、SEV和相关蛋白的稳定供应。b) 产生针对存在于外部结构域的多肽的多克隆抗体 激动素受体的表达及大鼠抗体的制备和纯化 腺激肽释放酶和颌下酶缩血管。 抗体将用于执行或发展RIA，并用于 免疫细胞化学定位研究。二)：维持杂交瘤和 制备和纯化抗蛋白质和蛋白质的单抗和Fab片段 根据不同项目调查人员的要求提供多肽。我们会 维持针对激动素、心钠素和大鼠的单抗供应 腺激肽释放酶，提纯免疫球蛋白并产生相应的 FAB碎片。抗体将通过使用 生物反应器技术。三)采用反相和凝胶排除高效液相色谱法 鉴定和分析多肽的技术。四)：执行分析和 参赛者需要的定量分子生物学技术 调查人员。需要聚丙烯酰胺凝胶电泳的技术， 核心还将进行印迹和放射自显影。V)：a) 在培养中维持已建立的细胞系。细胞系，如M1和 牛肺动脉内皮细胞，将按需要进行维护 由参与者调查人员提供。六)：执行分析技术，如 酶活力、蛋白质、电解质、菊粉和p- 氨基马尿酸盐。项目调查人员之间的密切沟通 生物化学核心人员将允许进行有效的规划，并允许更好地 协调研究工作。方法论的可变性将是 最大限度地减少，生物数据的整合将得到加强。在这 我们将通过这种方式提高效率，降低成本。