影响磁珠共价偶联效率的因素分析

目的研究不同条件下Dynabeads M-280 Tosylactivated磁珠与牛血清白蛋白(BSA)的偶联效率,探讨其影响因素。方法分别取Dynabeads M-280 Tosylactivated磁珠与BSA混合,在不同的pH条件、不同的硫酸铵浓度和不同的BSA浓度下37℃振荡孵育18 h,然后置于磁力架上静置2～3 min分离上清液。应用Bradford蛋白定量法对偶联前后的上清液进行蛋白定量分析,计算不同条件下的偶联效率。结果当缓冲液pH值为9.5时偶联效率为1.12μgBSA/mg磁珠,pH值为7.4时偶联效率为0.41μg BSA/mg磁珠;当反应体系中硫酸铵终浓度为1.2 mol/L时偶联效率为9.5μg BSA/mg磁珠,硫酸铵浓度为0.8 mol/L时偶联效率为8μg BSA/mg磁珠;当偶联的BSA终浓度为0.4 mol/L时偶联效率为9.5μg BSA/mg磁珠,BSA浓度为0.6 mol/L时偶联效率为13μg BSA/mg磁珠。结论缓冲液pH值、硫酸铵浓度和BSA的浓度会影响Dynabeads M-280 Tosylactivated磁珠与BSA的偶联效率,该偶联过程在pH9.5、1.2 mol/L硫酸铵浓度和0.6 mol/L BSA的条件下进行较为适合。

The influence of different experimental conditions on covalent coupling efficiency of magnetic beads

Objective To investigate covalent coupling efficiency of Dynabeads M - 280 Tosylactivated magnetic beads with bovine serum albumin （BSA） under different experimental conditions, and explore the influencing factors. Methods Dynabeads M -280 Tosylactivated mag- netic beads with BSA solution were mixed. The operation were performed separately in different pH buffer solutions, different concentrations of am- monium sulfate, and different concentrations of BSA. The tubes were placed on a magnet, allowing supernatants and magnetic beads to separate af- ter being incubated on a roller at 37~C for 18 hours. Bradford assay was applied to determine the concentrations of supernatants before and after the coupling. Subsequently, the coupling effieiencies of magnetic beads with BSA were figured out. Results When the experiment was conducted in pH 9.5 condition, the coupling efficiency was 1.12 ？ g BSA per mg magnetic beads, while the coupling efficiency was O. 41 ？ g BSA per mg magnetic beads in pH 7.4 condition. Adjusting the final concentration of ammonium sulfate in reaction system to 1.2 mol/L and 0.8mol/L sepa- rately, the corresponding coupling efficiencies were 9.5 ？ g BSA per mg magnetic beads and 8 ug BSA per mg magnetic beads. Adjusting the final concentration of BSA in reaction system to 0.4 moL/L and 0.6moL/L separately, the corresponding coupling efficiencies were 9.5 .9 g BSA per mg magnetic beads and 13 ug BSA per mg magnetic beads. Conclusion pH value of buffer solutions, as well as the concentration of ammonium sul- fate and BSA, can affect the coupling efficiency. Covalent coupling will proceed better in the condition of pH 9.5, 1.2 mol/L ammonium sulfate and O. 6 mol/L BSA .