【摘要】 目的:观察不同细胞裂解液对Bradford法和bicinchoninic acid(BCA)法的影响,以确定适合蛋白质组学研究中细胞全蛋白含量的定量方法。方法:采用Bradford法和BCA法测定牛血清白蛋白(bovine serum albumin,BSA)样品溶液,样品BSA溶液分别由双蒸水和不同裂解液(荧光差异双向凝胶电泳裂解液、三种传统双向凝胶电泳裂解液)配制;然后,采用这两种方法测定上述裂解液所提取的细胞全蛋白样品;采用Bradford法定量反复冻融、不同比色杯和不同时期标准曲线下的样品。结果:各细胞裂解液对Bradford法均有显著性影响,使所定量的BSA浓度普遍偏高1.2至2倍,但定量值可随着加样浓度的增加而增加(r=0.989~0.996,P<0.05);各裂解液对BCA法也均有显著性的干扰,多数定量结果超出仪器读数范围;对于同批全蛋白溶液样品,BCA法的定量结果与Bradford法相比可有千倍差异;反复冻融后蛋白浓度变化统计学上无显著性差异,但有下降趋势,不同比色杯和不同时期标准曲线对Bradford法无显著影响。结论:Bradford法是蛋白质组学中细胞全蛋白定量的较适方法,但需根据具体实验进行优化。更多还原

【Abstract】 Objective: To observe the effects of different cell lysis buffers on protein quantification with Bradford method and bicinchoninic acid(BCA) method.Methods: Bradford method and BCA method were used to determine the concentration of bovine serum albumin(BSA) in different solutions(distilled water,cell lysis buffer used in two-dimensional differential in-gel electrophoresis and three kinds of cell lysis buffers used in conventional two dimensional gel electrophoresis),as well as the protein concentrations of cell lysates using these different lysis buffers.Bradford method was also applied to determine the protein concentrations of samples with repeated freeze thaw cycle,in different colorimetric cylinders,or using different standard curves from different periods.Results: The protein measurements increased for 1.2 to 2 fold when different cell lysis buffers were used in Bradford method,but the measurements increased with the increased concentration of BSA(r=0.989~0.996,P<0.05).For BCA,measurement reading increased about thousands times higher,even overflowed the limits of machine.Protein measurements didn’t change significantly,only showed a declined trend after repeated freeze thaw cycle,while no significant changes were found using different colorimetric cylinders or standard curves from different periods.Conclusions: Bradford method may be the choice of the protein quantification in proteomics.However,optimization is required for specific experimental conditions.更多还原