Turbidity Assays of Liquid-Liquid Phase Separation

Liquid-liquid phase separation (LLPS) results in the formation of two distinct phases, containing a dilute phase and a dense phase. As the solution undergoing LLPS forms tiny droplets, the turbidity of the solution is altered. Thus, if phase separation of the protein occurs, the solution becomes milky or cloudy in appearance; otherwise, it remains clear. Mesoscale components in solution with diameters on the order of tens to hundreds of nanometers (nm) scatter visible light and can be detected by optical density measurements (typically at 340 nm or 400 nm) or by direct static light scattering.

Fig. 1. Working principle of turbidity assay. (Huang Y, et al., 2021)

Customized Services

Here, we use fixed wavelength optical density measurements to detect turbidity measurements performed on LLPS solutions. CD BioSciences offers turbidity meters, nephelometers, or spectrophotometers to determine the phase boundaries of protein and/or RNA systems by detecting changes in the turbidity of the solution. We measure turbidity according to the gradient of protein concentration of your project to detect the onset of turbidity, which is observed as a measurement of the saturation concentration of LLPS.

Turbidity measurements provide only a preliminary LLPS study that can detect multiple combinations.

• Combined with complementary imaging studies to assess the physical properties of phase separators.
• Combined with UV absorbance measurements to quantify the concentration of components in the concentrated and dilute phases after partitioning by centrifugal distribution. We are proud to offer UV-Vis spectrophotometric turbidity measurements to characterize the temperature and concentration dependence of protein LLPS.
• Combined with automated liquid handling equipment and enzyme markers for high-throughput LLPS analysis. Increased solution turbidity can be attributed to a variety of phenomena, including phase separation into dense liquids, hydrogels or solid particles, or non-specific protein aggregation.
• Combined with structural domain deletion and targeted mutagenesis to help you analyze the structural features of proteins required for phase separation.

Advantages of Turbidity Assays for Liquid-Liquid Phase Separation

• Can be used to determine the concentration threshold for phase separation.
• The temperature dependence of phase separation can be monitored.
• Non-destructive.
• Suitable for high throughput.

Our turbidimetric services are widely used for liquid-liquid phase separation (LLPS) of proteins in in vitro chemical analysis. Faced with the common pitfalls of this assay due to being outside the linear range of detection, our experts explore simple sample dilution of insoluble aggregated protein samples or direct imaging of droplets to address these issues and improve the accuracy of turbidimetric assays. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.