摘要
增强型绿色荧光蛋白(EGFP)是生物领域常用的标记物。在前期成功克隆表达EGFP的基础上,本实验建立了两步分离纯化EGFP的色谱方法,并验证其分离纯化效果,检验EGFP的活性。首先用金属螯合亲和色谱柱His-Trap HP对EGFP的重组菌体破碎上清液进行初步分离,再用葡聚糖凝胶排阻色谱柱Sephadex G-10 HR对其进行脱盐纯化。采用丙烯葡聚糖凝胶排阻色谱柱Sephacryl S-300 HR和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测分离纯化后的EGFP纯度。最后通过荧光分光检测器和非变性聚丙烯酰胺凝胶电泳(Native-PAGE)验证分离纯化后的EGFP是否具有荧光活性。结果表明该方法可以简便快速地分离纯化EGFP,纯度超过98%,同时保持了EGFP的荧光活性。
Enhanced green fluorescent protein （EGFP） is a common biological marker. In this research, on the foundation of successful clone and expression of EGFP, a two-step chromatographic method was established to separate and purify EGFP, which includes the use of His- Trap HP immobilized metal affinity chromatography （IMAC） and Sephadex G-10 HR size exclusion chromatography in sequence. Sephacryl S-300 HR size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） were used to check out the purity of EGFP. At last, it was found that EGFP still had fluorescent activity using fluorescence spectrophotometric detection and Native-PAGE detection. This method can effectively separate the active EGFP. The purity of the obtained EGFP was more than 98%.
出处
《色谱》
CAS
CSCD
北大核心
2013年第2期151-154,共4页
Chinese Journal of Chromatography
关键词
金属螯合亲和色谱
凝胶排阻色谱
增强型绿色荧光蛋白
分离
纯化
immobilized metal affinity chromatography （IMAC）
gel exclusion chromatography （ GEC ）
enhanced green fluorescent protein （EGFP）
separation
purification
