基于非对称流场流分离技术的蛋白分离研究

Protein separation based on asymmetric flow field-flow fractionation

通过对肌红蛋白和牛血清白蛋白混合蛋白样品分离,研究了非对称流场流分离技术(AF4)分离蛋白时需要优化的参数及各个参数对分离效果的影响。首先应用正交设计实验考察样品进样量、横向流速、进检测器流速、聚焦时间对分离效果的影响,得到最大影响因素为横向流速,最优分离条件为:样品进样量20μL、横向流速5mL/min、进检测器流速0.2mL/min、聚焦时间6min。经验证此最优分离条件下分离度达到2.5。随后在最优分离条件下分别考察膜材料、缓冲盐浓度、分离腔室厚度对非对称流场流分离效果的影响。结果表明,截留膜选用再生纤维素膜,流动相缓冲盐浓度为20mmol/L时,满足非对称流场流分离技术分离蛋白样品的分离要求,随着腔室厚度的提高,分离度进一步增大。

The effect of varying the experimental parameters on asymmetrical flow field-flow fractionation(AF4)has been studied by separating a mixture of two proteins(myoglobin and bovine serum albumin).Firstly,the effects of injection volume,cross flow rate,detector flow rate,and focusing time on the resolution were investigated by orthogonal tests.The cross flow rate was shown to be the factor with the greatest impact,and the optimal separation conditions were found to be asample injection volume of 20μL,a cross flow rate of 5mL/min,a detector flow rate of 0.2mL/min,and a focusing time of 6min.The resolution was 2.5 under the optimal separation conditions.The influence of the membrane material,buffer concentration and channel spacer on the AF4 separation of proteins was studied under the optimal separation conditions.The results showed that a regenerated cellulose membrane and a mobile phase buffer concentration of 20mmol/L enabled separation of the proteins,and the separation efficiency could be improved byincreasing the channel spacer.