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 是分离出足够数量的纯物种，并保留 本机配置。反相高压液相色谱 常规和窄孔格式和十二烷基硫酸钠的电泳法 缓冲体系后的电洗脱或电吸印分离 组件代表了最有效的方法，无论何时亲和性或 其他凝胶层析技术不适用。然而，许多人 感兴趣的大分子不适合这些方法，因为低 在存在的情况下分离期间生物活性的恢复和损失 有机溶剂或十二烷基硫酸钠。我们正在开发 多相区带法纯化生物活性分子技术 非变性条件下的聚丙烯酰胺凝胶电泳法 结合高效毛细管电泳法和精心控制温度下的洗脱 和缓冲剂组合物。缓冲系统被设计成包含 手术的酸碱值在7.5到11.0之间，并为 具有酸性到碱性等电点的蛋白质。高分辨率 聚丙烯酰胺凝胶电泳力和分离能力 根据分子的大小、净电荷和 疏水性将应用于分离天然细胞因子，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 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 represented 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.