Process Biochemistry 46 (2011) 413–417 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Short communication Colorimetric protein quantification in aqueous two-phase systems Mirna González-González, Karla Mayolo-Deloisa, Marco Rito-Palomares, Robert Winkler ∗ Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico article info Article history: Received 18 December 2009 Received in revised form 8 June 2010 Accepted 31 August 2010 Keywords: Protein quantification Aqueous two-phase systems Colorimetric protein assay abstract The polymer-salt rich environment of aqueous two-phase systems disturbs standard protein quantifica- tion 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. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Aqueous two-phase systems (ATPS) are applied for the capture and partial purification of proteins from crude feedstocks. In com- parison to other primary downstream strategies, ATPS processes are characterized by high capacity, comparatively low costs and ease of scale up. Moreover, the required time for phase separation is relatively short and the mild separating conditions help maintain- ing biological activities [1–3]. ATPS are composed of two immiscible aqueos solutions. For preparative scale, ATPS consisting of PEG and phosphate salts have reached by far the highest importance due to their economy and pH stability [4]. The development and broad application of ATPS is hindered by two restrictions. Firstly, a reliable prediction for the phase behavior of a specific protein in a given ATPS is not possible yet. Secondly, monitoring the fate of proteins–a prerequisite for evalu- ating bioprocesses - is frequently disturbed by the presence of high concentrations of polymers or salts (up to 30% are common) [5,6]. One of the most important methods for protein quantifica- tion is the measurement of the optical absorbance, typically at a wavelength of 280 nm. This method is highly accurate, linear in Abbreviations: ATPS, aqueous two-phase systems; BSA, bovine serum albumin; PEG, polyethylene glycol; RNase A, ribonuclease A. ∗ Corresponding author. CINVESTAV Unidad Irapuato, Km. 9.6 Libramiento Norte Carr. Irapuato-León, 36821 Irapuato Gto., México. Tel.: +52 462 6239 635. E-mail address: robert.winkler@ira.cinvestav.mx (R. Winkler). a certain range and can be performed online [8–11]. However, the UV absorption coefficient is protein specific and the measurements can be falsified by compounds present in the analyte solutions. In a recent study we could determine absorption coefficients between 0.4157 and 1.3273 ml mg -1 cm -1 for RNase A, depending on the phase composition of ATPS solutions [7]. Consequently, a number of calibration curves would be necessary to account for different com- binations of protein, salt and polymer content. Such a tedious and time-consuming procedure is not feasible for screening of many different ATPS conditions. The other major strategy for protein determination in biosep- aration fractions is based on colorimetric assays, which estimate the protein concentration through the interaction of the protein or amino acids with a colorant, resulting in changes of the optical absorbance. These optical changes are quantified by photometry and in comparison with standard curves the protein concentration can be determined. Colorimetric assays offer the possibility to per- form microplate tests, thus reducing the required sample volume and increasing the number of samples assayed in parallel. Addition- ally they display high sensitivity, accuracy and reproducibility and are easy to perform with low investments for necessary equipment [6,13]. The colorimetric protein assays selected for this study are the Bradford assay, the Bio-Rad DC assay and the ninhydrin assay. The Bradford protein assay relies on the adsorptive binding of Coomassie Brilliant Blue G250 to protein [8]. The anionic form of the dye binds to cationic and non-polar amino acid residues of the proteins, preferably on arginine residues. This Coomassie-protein complex absorbs strongly at 595 nm [9]. 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.08.026