Date Published: April 19, 2019
Publisher: Public Library of Science
Author(s): Zsolt Datki, Zita Olah, Lilla Macsai, Magdolna Pakaski, Bence Galik, Gabor Mihaly, Janos Kalman, Aldrin V. Gomes.
In many biology- and chemistry-related research fields and experiments the quantification of the peptide and/or protein concentration in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different experiments beyond or instead of the well-known standard protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This molecule has several advantageous properties related to protein detection: good solubility in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold diluted samples) concentrations (from 0.28 up to more than 100 μg/mL). The BisANS protein assay is valid and applicable for quantification of the amount of protein in different biological and/or chemical samples.
Accurate peptide and/or protein quantification is essential in a multitude of research topics. Different methods were developed to measure the amount of proteins originating from various types of biological samples. A majority of them are fluorescence- (e.g. Qubit) or absorbance-based assays, such as the traditional Coomassie blue G-250 dye-binding  (Bradford) and the bicinchoninic acid (BCA)  assay. Both the Bradford and the BCA assays are based on color change in the visible spectrum as a response to the presence of proteins. The color formation observed in the Bradford assay is a result of complex formation between proteins and the Coomassie blue G-250 dye through electrostatic and hydrophobic interactions, where the anionic blue form of the dye is stabilized and could be measured . The BCA assay is based on the reduction of Cu2+ to Cu1+ by protein in an alkaline medium. Cu1+ forms a complex with BCA, resulting a colored water-soluble chelate . The intensity of the color change by these assays is measured by absorbance photometry at 595 nm and 562 nm for the Bradford and BCA assays respectively . Both methods allow the detection of proteins in μg/mL range. However, every assay has its own specific limitation and unique requirements (different incubation times, stabilizations, metal ions, pH, photosensitivity, chelator- and detergent sensitivity) .
Different methods are used to quantify the total amount of protein in a multitude of research topics; however, each assay has its own limitations (e.g. sensitivity to chelators and detergents). Due to the development of high throughput proteomic screening, there is an increasing demand for developing new, easy-to-use approaches. The BisANS fluorescent dye has been introduced in several fields of protein analysis. To characterize our novel BisANS-based protein quantification assay we tested it with protein samples prepared in basic medium (Fig 1) or in lysis buffer (Fig 2). First of all, we recommended an optimal incubation time and environment for this assay using BSA as standard in chelator, detergent and inhibitor free basic medium. To reach the highest reliability, the relative fluorescence intensity, and the recommended incubation time were indicated (Fig 1A). We suggest subjectively 120 sec incubation time for saturation of fluorescent signal; however, we believe that it is unnecessary to wait any longer. The photostability of the BisANS was tested under different light conditions: dark, white (400 and 40,000 lux) and ultraviolet (UV; 254 and 366 nm) ones (Fig 1B). No significant changes were observed. The concentration range and minimum detection limit were evaluated by BSA standard ranging from 0 to 100 μg/mL, and the correlation coefficient (R2 = 0.9927; y = 112.64x + 203.27) was calculated (Fig 1C). The minimum detection limit was 0.65 μg/mL. To validate our assay, Bradford and BCA methods were used, paralelly measuring known (BSA) and unknown (NCS) protein concentrations. We found that the BisANS assay proved to be the most accurate (Fig 1D).