河北科技大学学报2025，Vol.46Issue(5)：553-560,8.DOI:10.7535/hbkd.2025yx05008
考马斯亮蓝法测定蛋白质含量的优化
Optimization of Coomassie Brilliant Blue method for determination of protein content
摘要
Abstract
This study addressed the issues of insufficient sensitivity and poor stability of traditional Coomassie Brilliant Blue method in trace protein detection by systematically optimizing each procedural step based on its reaction principles.Firstly,a microplate reader combined with 96-well plates was employed to replace traditional ultraviolet spectrophotometers and cuvettes,thereby improving detection throughput and precision.In addition,20 mmol/L phosphate buffer(PB)was used as the diluent instead of water to enhance protein solubility.Lastly,the optimal formulation of Coomassie Brilliant Blue G-250 solution was prepared by dissolving 5 mg Coomassie Brilliant Blue G-250 in an aqueous solution containing 5%of 95%ethanol and 13%of 85%phosphoric acid.Validation results demonstrate that,the optimal reaction system involved an equal volume of bovine serum albumin(BSA)mixed with the Coomassie Brilliant Blue G-250 solution,with the ideal reaction conditions being a 10-minute incubation in the dark.The method demonstrates excellent linearity(R2>0.99)within the protein concentration range of 0～21 mg/L,with a detection limit as low as 1 μg/mL.Both satisfactory accuracy(80%～115%)and precision(RSD<4%)meet pharmacopoeia reference standards.This study successfully achieves accurate quantitative analysis of trace proteins,offering a cost-effective,rapid,and high-throughput approach,thereby providing more precise and efficient technical support for protein-related research.关键词
应用生物化学/蛋白质含量测定方法/考马斯亮蓝/稳定性/检测限Key words
applied biochemistry/method for determination of protein content/Coomassie Brilliant Blue/stability/detection limit分类
生物学引用本文复制引用
刘金铭,李丽,王培,刘力强,刘浩,周晓辉..考马斯亮蓝法测定蛋白质含量的优化[J].河北科技大学学报,2025,46(5):553-560,8.基金项目
国家药典委员会国家药品标准提高项目(2021Y04) （2021Y04）
河北省外专百人计划项目(2200626) （2200626）
河北省农业科技成果转化资金项目(202460101010040) （202460101010040）
