Field Nicholas, Konstantinidis Spyridon, Velayudhan Ajoy
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom.
J Chromatogr A. 2017 Aug 11;1510:13-24. doi: 10.1016/j.chroma.2017.06.012. Epub 2017 Jun 6.
The combination of multi-well plates and automated liquid handling is well suited to the rapid measurement of the adsorption isotherms of proteins. Here, single and binary adsorption isotherms are reported for BSA, ovalbumin and conalbumin on a strong anion exchanger over a range of pH and salt levels. The impact of the main experimental factors at play on the accuracy and precision of the adsorbed protein concentrations is quantified theoretically and experimentally. In addition to the standard measurement of liquid concentrations before and after adsorption, the amounts eluted from the wells are measured directly. This additional measurement corroborates the calculation based on liquid concentration data, and improves precision especially under conditions of weak or moderate interaction strength. The traditional measurement of multicomponent isotherms is limited by the speed of HPLC analysis; this analytical bottleneck is alleviated by careful multivariate analysis of UV spectra.
多孔板与自动化液体处理相结合非常适合快速测量蛋白质的吸附等温线。本文报道了牛血清白蛋白（BSA）、卵清蛋白和伴清蛋白在强阴离子交换剂上，在一系列pH值和盐浓度条件下的单组分和双组分吸附等温线。从理论和实验两方面对影响吸附蛋白质浓度准确性和精密度的主要实验因素进行了量化。除了对吸附前后液体浓度进行标准测量外，还直接测量了从孔中洗脱的量。这一额外测量证实了基于液体浓度数据的计算，并提高了精密度，特别是在相互作用强度较弱或中等的条件下。传统的多组分等温线测量受高效液相色谱（HPLC）分析速度的限制；通过对紫外光谱进行仔细的多变量分析，缓解了这一分析瓶颈。
