ISOLATION OF BIOLOGICALLY ACTIVE CYTOKINES BY NON-DENATURING PAGE

生物大分子研究中最困难的步骤之一 分离出足够量的纯物质， 本机配置。 反相高压液相色谱 以常规和窄孔形式和SDS中电泳 缓冲液系统，然后进行电洗脱或电印迹， 组件代表最有效的方法，无论何时， 其它凝胶色谱技术不适用。 但不少 感兴趣的大分子不适合这些方法， 在分离过程中生物活性的低回收和损失 存在有机溶剂或十二烷基硫酸钠。 我们 开发技术来纯化生物活性分子， 聚丙烯酰胺凝胶多相区带电泳 非变性条件，结合HPEC和洗脱条件， 仔细控制温度和缓冲液组成。 缓冲 系统被设计为包括7.5至11.0的操作pH值 并为酸性到碱性的蛋白质提供足够的流动性 等电点 聚丙烯酰胺凝胶的高分辨率 电泳和分离分子的能力， 尺寸、净电荷和疏水性的差异将被应用于 天然细胞因子、人甲状腺肿蛋白和破伤风毒素的分离， 和重组干扰素-α受体。

One of the most difficult steps in the study of biological macromolecules is the isolation of sufficient amounts of pure species with retention of native configuration. Reverse phase high pressure liquid chromatography in both conventional and narrow bore format and electrophoresis in SDS buffer systems followed by electroelution or electroblotting of resolved components represent the most efficient methods whenever affinity or other gel chromatography techniques are not applicable. However, many macromolecules of interest are not amenable to these approaches due to low recovery and loss of biological activity during separation in the presence of organic solvents or sodium dodecyl sulfate. We are developing techniques to purify biologically active molecules by multiphasic zone electrophoresis in polyacrylamide gel under non-denaturing conditions, in conjunction with HPEC and elution under carefully controlled temperature and buffer composition. The buffer systems are designed to encompass operative pH's ranging from 7.5 to 11.0 and to provide enough mobility for proteins with acidic to basic isoelectric points. The high resolving power of polyacrylamide gel electrophoresis and the ability to separate molecules on the basis of differences in size, net charge and hydrophobicity will be applied to the isolation of native cytokines, human goiter proteins and tetanus toxins, and recombinant interferon-alpha receptor.