Continued advancement of protein array, bioelectrode, and biosensor technologies will necessitate development of methods that allow for increased protein immobilization capacity and more control over protein orientati...Continued advancement of protein array, bioelectrode, and biosensor technologies will necessitate development of methods that allow for increased protein immobilization capacity and more control over protein orientation. Toward these ends, we developed a method involving modification of chitosan with nitrilotriacetic acid (NTA) to achieve immobilization of a larger amount of His-tagged protein than is possible with current methods. The immobilization capacity of our method was evaluated using His-tagged GFP (Green Fluorescent Protein) as a model protein. The average immobilization density on modified glass was about 32 ng/mm2. Our method is suitable for use on a variety of solid surfaces, including glassy carbon, silicon wafers, polycarbonate, and beaten gold.展开更多
目的:提纯基因工程菌1020耐热木聚糖酶。方法:超声破碎细胞,而后经过热变性处理和Ni-NTA亲和层析分离纯化。结果:根据pET System Manual的方法分析木聚糖酶主要分布在可溶性细胞质中;超声破壁功率400W,破碎15min时效果最佳;粗酶液经过70...目的:提纯基因工程菌1020耐热木聚糖酶。方法:超声破碎细胞,而后经过热变性处理和Ni-NTA亲和层析分离纯化。结果:根据pET System Manual的方法分析木聚糖酶主要分布在可溶性细胞质中;超声破壁功率400W,破碎15min时效果最佳;粗酶液经过70℃,热变性30min处理后,纯化倍数达到4.9;采用Ni-NTA Sephrose F.F一步层析可以得到电泳纯的木聚糖酶,纯化倍数13.4,得率29.4%。结论:热变性处理是一种有效的提纯手段,合理的条件可以使纯化倍数提高;Ni-NTA亲和层析是纯化含His标签的目的酶的特异高效纯化方法。展开更多
文摘Continued advancement of protein array, bioelectrode, and biosensor technologies will necessitate development of methods that allow for increased protein immobilization capacity and more control over protein orientation. Toward these ends, we developed a method involving modification of chitosan with nitrilotriacetic acid (NTA) to achieve immobilization of a larger amount of His-tagged protein than is possible with current methods. The immobilization capacity of our method was evaluated using His-tagged GFP (Green Fluorescent Protein) as a model protein. The average immobilization density on modified glass was about 32 ng/mm2. Our method is suitable for use on a variety of solid surfaces, including glassy carbon, silicon wafers, polycarbonate, and beaten gold.
文摘目的:提纯基因工程菌1020耐热木聚糖酶。方法:超声破碎细胞,而后经过热变性处理和Ni-NTA亲和层析分离纯化。结果:根据pET System Manual的方法分析木聚糖酶主要分布在可溶性细胞质中;超声破壁功率400W,破碎15min时效果最佳;粗酶液经过70℃,热变性30min处理后,纯化倍数达到4.9;采用Ni-NTA Sephrose F.F一步层析可以得到电泳纯的木聚糖酶,纯化倍数13.4,得率29.4%。结论:热变性处理是一种有效的提纯手段,合理的条件可以使纯化倍数提高;Ni-NTA亲和层析是纯化含His标签的目的酶的特异高效纯化方法。
