ISOLATION OF BIOLOGICALLY ACTIVE CYTOKINES BY NON-DENATURAING PAGE

生物大分子研究中最困难的步骤之一 分离足够量的纯物种， 本机配置。反相高效液相色谱法 常规和窄孔形式以及SDS缓冲液中的电泳 系统，然后进行电洗脱或电印迹， 组件代表了最有效的方法， 其它凝胶色谱技术不适用。但不少 感兴趣的大分子不能用于这些方法， 生物活性的恢复和丧失 有机溶剂或十二烷基硫酸钠。我们正在开发技术， 通过多相区带电泳纯化生物活性分子， 聚丙烯酰胺凝胶在非变性条件下，结合 在仔细控制的温度和缓冲液下进行HPEC和洗脱 混合物.缓冲系统设计为包括操作pH值 范围从7.5到11.0，并为蛋白质提供足够的流动性， 酸性到碱性等电点。高分辨率的 聚丙烯酰胺凝胶电泳和分离分子的能力 大小、净电荷和疏水性差异的基础将是 应用于天然细胞因子、人甲状腺肿蛋白和 破伤风毒素

One of the most difficult steps in the study of biological macromolecules is he 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 represented the most efficient methods whenever affinity or other gel chromatography techniques are not applicable. However, many macromolecules of interest are not enable to these approaches due to low recovery and loss of biological activity ring 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.