Quantitation of nucleoprotein complexes by UV absorbance and Bradford assay

Abstract

Despite the importance of studying nucleoprotein complexes, no appropriate method for quantifying them is available. Here, a UV absorbance method using the formula “C_mg/mL = 1.55A₂₈₀ – 0.76A₂₆₀” were applied to quantify nucleoprotein complexes. After modification using two paired A₂₆₀ and A₂₈₀ values, the UV-derived formula-based method could accurately quantify proteins in nucleoprotein complexes. Otherwise, by taking the target protein as a standard, the Bradford assay can accurately quantify proteins in nucleoprotein complexes without interference by nucleic acids. The above methods were successfully applied to measure the concentration of MtuP49-CTG complexes ofMycobacterium tuberculosis. In conclusion, both the Bradford assay and the UV-derived formula-based method were appropriate for quantifying proteins in nucleoprotein complexes, which may make contributions to explore the interactions between proteins and nucleic acids at the molecular level.

Graphical abstract

Keywords

Nucleoprotein complex / Quantitation / Bradford assay / UV-derived formula-based method

Funding

This work was supported by grants from the National Natural Science Foundation of China (31470742, 31700664, U1432102, 31270783), an initial grant from the 100 Talents Program of the Chinese Academy of Sciences, and the Key Laboratory of Environmental and Applied Microbiology at the Chinese Academy of Sciences. We thank Dr. Aparicio for the gift of the Fhk1-DBD plasmid.