The use of antifoam agents to eliminate bubbles during biotechnological sample analysis

Abstract

Colorimetric protein quantitation assays such as the Bradford method are susceptible to foaming when using manual or automated equipment, especially during mixing operations. The resulting bubbles cause unintended light scattering and, because they often persist for longer than the duration of the assay, can interfere with spectrometric sample analysis, for example in a 96-well plate reader at 585 nm. Here, we tested the ability of antifoam agents commonly used in biotechnology to reduce bubble formation when mixing Bradford reagent with protein samples from bioprocess development and production. We also assessed the impact of these agents on optical density at the assay-relevant wavelength to verify the fidelity of the readouts. We used a design of experiments approach to identify synergies between different antifoam agents, aiming to reduce the concentration needed to avoid foam formation and thus minimize any distortion of the assay results. Finally, we confirmed that antifoam-containing Bradford reagent retained its performance even after prolonged storage. We found that 0.15 g L−1 of Struktol J673A was sufficient to prevent foaming even when air was deliberately introduced into the samples. Furthermore, the modified Bradford reagent was stable for 4 weeks and did not distort the readout at 585 nm but only shifted it by ~ 0.1 dimensionless absorbance units. Therefore, our modified Bradford reagent will benefit biologists, biotechnologists and bioprocess engineers, especially those using automated workstations, because it improves the robustness of the assay.