Determination of Diffusivity of Biomolecular Condensates

A large number of membraneless organelles in cells are assembled by liquid-liquid phase separation (LLPS). The diffusion coefficients of the components in the dilute phase of these condensates are usually high and decrease gradually in the dense phase as the state of matter changes from liquid to hydrogel to solid. The diffusion coefficient of dense phase components reflects the mobility of biomolecular condensates and is a direct parameter to characterize the state of matter of biomolecular condensates. Different condensates contain components with different diffusion coefficients to achieve different biological functions. Dysfunctional or abnormal changes in the diffusivity of the components of biomolecular condensates disrupt its physiological kinetics and function. Such alterations are associated with the pathogenesis of several diseases, such as the coagulation of multiple RNP particles in several neurodegenerative diseases.

Customized Services

The diffusivity of different components of the same condensate varies widely, and the different diffusivity of these different components is important for their physiological function. Our team of experts is dedicated to measuring the diffusivity of multiple individual components to accurately characterize the state of matter of biomolecular condensates. Here, CD BioSciences offers the fluorescence recovery after photobleaching (FRAP) method to assess condensate mobility and estimate protein diffusion coefficients.

We provide detailed procedures to probe the diffusivity of fluorescently labeled protein/RNA molecules in biomolecular condensates in vitro and in vivo.

(1) Sample preparation. Condensate samples containing fluorescently labeled species are placed on Tween-coated microscope slides/coverslips.

(2) Fluorescence imaging. Take a fluorescence image of the sample on the glass surface to ensure that the labeled molecules are being distributed into the droplet and that the fluorescence intensity is appropriate. Adjust the laser power and/or detection settings to ensure that the appropriate level of sample brightness is well within the dynamic range of the detector.

(3) Focus the confocal volume on an area where you can see at least two droplets fixed on the surface of the slide.

(4) Set the bleaching point at the center of one of the droplets, ensuring that the size of the bleaching point is significantly smaller than the size of the droplet, thereby improving the accuracy of the analysis and minimizing interfacial resistance effects.

(5) Photobleaching. Irradiate the excitation laser with maximum intensity for 1-2 seconds to start the bleaching experiment.

(6) Imaging of two droplets after a fixed time interval of bleaching.

(7) Data analysis and statistics.

In addition, we combine the microfluidic device with particle tracking method to analyze rheological properties and measure diffusion coefficients.

Advantages of FRAP Method

• A fast and simple method to obtain the diffusivity dynamics of fluorescently labeled molecules in biomolecular condensates.
• Easy to implement in in vitro and cell culture models.
• Does not require specialized equipment.
• Allows preliminary determination of whether the condensate is a liquid or a solid.
• 5 μL of sample is sufficient for >30 min imaging.

CD BioSciences can precisely determine the diffusivity within biomolecular condensates by FRAP experiments, one of the few molecular diffusivity experiments that can be performed in vivo on biomolecular condensates. We demonstrate the accuracy and precision of our method by considering spatially and temporally resolved data for protein condensates and two different polyelectrolyte condensate layer systems. 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.