Colorimetric protein quantification in aqueous two-phase systems

The polymer-salt rich environment of aqueous two-phase systems disturbs standard protein quantification methods, like UV measurement or Bradford assay. Therefore, we systematically investigated the influence of high polyethylene glycol and phosphate salt concentrations on the readings of three colorimetric protein assays: Bradford, DC (Bio-Rad) and ninhydrin assay. All three assays are troubled by the aqueous two-phase systems components, but disturbances can be significantly reduced by diluting the samples to 1% PEG and 10% salt. But additionally, the Bradford and the DC assay are strongly dependent on the protein composition, which lead to analytical protein recoveries from 0% to 181% in the current study. Especially for salt-rich fractions with low protein concentration and for measuring analytically problematic proteins like RNase A we therefore suggest a ninhydrin assay, which displays minimal protein-to-protein variation and high sensitivity. (C) 2010 Elsevier Ltd. All rights reserved.