以3.0μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋...以3.0μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观解离常数为5.26×10-5mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。展开更多
目的:建立一种简便快捷的分离纯化肾病患者尿IgG亚类的方法。方法:收集的肾病患者24h尿液经硫酸铵盐析、DEAE-cellu lose离子交换层析和葡萄球菌A蛋白(SPA)亲和层析等3步分离纯化,即可得到IgG1、IgG2、IgG3、IgG44种亚类,最后用W estern...目的:建立一种简便快捷的分离纯化肾病患者尿IgG亚类的方法。方法:收集的肾病患者24h尿液经硫酸铵盐析、DEAE-cellu lose离子交换层析和葡萄球菌A蛋白(SPA)亲和层析等3步分离纯化,即可得到IgG1、IgG2、IgG3、IgG44种亚类,最后用W estern b lotting和SDS-PAGE电泳的方法鉴定样品及其纯度。结果:SPA亲和层析洗脱曲线显示有4个独立的洗脱峰,W estern b lotting和SDS-PAGE方法鉴定表明提纯的样品为高纯度的IgG1、IgG2、IgG3、IgG4。结论:本方法通过3步即可一次性将样品中的4种不同的IgG亚类全部分离纯化,且纯度高,简便快捷,效果良好。展开更多
Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their ...Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.展开更多
文摘以3.0μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观解离常数为5.26×10-5mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。
文摘目的:建立一种简便快捷的分离纯化肾病患者尿IgG亚类的方法。方法:收集的肾病患者24h尿液经硫酸铵盐析、DEAE-cellu lose离子交换层析和葡萄球菌A蛋白(SPA)亲和层析等3步分离纯化,即可得到IgG1、IgG2、IgG3、IgG44种亚类,最后用W estern b lotting和SDS-PAGE电泳的方法鉴定样品及其纯度。结果:SPA亲和层析洗脱曲线显示有4个独立的洗脱峰,W estern b lotting和SDS-PAGE方法鉴定表明提纯的样品为高纯度的IgG1、IgG2、IgG3、IgG4。结论:本方法通过3步即可一次性将样品中的4种不同的IgG亚类全部分离纯化,且纯度高,简便快捷,效果良好。
基金supported by grants from the Brazilian Agency Coordination of Graduate Level Training(CAPES,project 0366/09-9)State of So Paulo Research Support Foundation(FAPESP-Brazil,project 2005/60159-7)
文摘Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.
