关键词: 蛋白质组学, 双向电泳, Bradford法, 蛋白质定量, 蛋白质裂解液

Abstract: Bradford assay is the most widely used protein quantification method for its low cost, high sensitivity and rapid and simple determination. To enhance the suitability of the Bradford assay for proteomic research, the assay was optimized specifically for two-dimensional electrophoresis (2-DE) samples. Through constructing the correspondence of protein content (0~1 000 μg) with absorbance at 595 nm, the linearity range was determined at 0~100 μg. The interference of lysis buffer and its components, and different detergents was tested. Results showed that there was a positive correlation between interference of lysis buffer and sample volume. By analyzing the absorbance change at 595 nm of protein samples with or without lysis buffer, lysis buffer was found to have an effect on the stability of protein-dye complex and the proposed measurement time for 2-DE samples was between 3 and 30 min after mixing protein samples with dye reagent. Increasing in phosphoric acid concentration from 8.5% to 10.2% (w/V) in the dye reagent can improve the stability of protein-dye complex. As the absorbance of the same amount of protein was quite different whether or not lysis was present, the addition of corresponding volume of lysis buffer to the standard protein samples and the reagent blank must be ensured. Sample volume was adjusted from 100 μL to 10 μL. The adjustment of sample volume lowered fluctuation of measured data and improved linearity of the standard curve, as well as simplified the quantitative analysis by elimination of dilution process.

Key words: proteomics, two-dimensional electrophoresis, Bradford assay, protein quantification, lysis buffer