Phase Separation Analysis of Purified Proteins

Cells contain many membrane-free compartments formed by phase separation. They typically have the properties of droplets and exchange components rapidly with their surroundings. In vitro phase separation assays with purified proteins have become the standard method for studying proteins that form membrane-free compartments. Various proteins have been purified and tested for their ability to phase separate and form droplets. However, the experimental results are somewhat variable and not easily reproducible. Also many phase separated proteins are difficult to purify and handle. Currently, the most commonly used phase separation protein expression system is bacterial. However, this expression system is limited by the fact that many phase isolated proteins have complex structural domain organization, are modified by post-translational modifications, and are highly aggregated in bacterial expression systems.

Fig. 1. A user's guide for phase separation assays with purified proteins. (Alberti S, et al., 2018)

Customized Services

Phase-separated proteins have unique structural features that allow them to self-interact and even aggregate at high concentrations. Many standard purification strategies are unable to purify phase-separated proteins. CD BioSciences offers corresponding strategies to optimize phase-separated protein purification.

• Enhancing the Solubility of Phase-Separated Proteins
  We screen for conditions that enhance protein solubility and place these tags near the region driving phase separation to interfere with self-assembly, thereby increasing solubility during purification.
• Reducing the Non-Specific Viscosity of Phase-Separated Proteins
  We use multi-step affinity chromatography with low binding resins and protease cleavage elution to reduce the non-specific stickiness of phase separated proteins.
• Selecting the Right Salt Concentration
  We typically use a high salt buffer during phase separation protein purification and perform it at room temperature.
• Selecting the Right Physicochemical Conditions
  Maintain dilution of the protein of interest during purification, select the appropriate buffer pH and add charged amino acids to the buffer to improve protein solubility.

CD BioSciences offers a customized process to achieve phase separation analysis of proteins in the following steps.

(1) Sample preparation. We induce phase separation by adding high quality proteins to the phase separation buffer, ensuring that the proteins are well mixed in the buffer.

(2) Imaging of the phase-separated condensates. We have an advanced inverted microscope platform to image the phase separated condensates. In addition, we use lipid bilayers or PEG-silane-coated coverslips or plate bottoms to preserve the material properties of the condensates, allowing for prolonged imaging.

(3) Monitoring kinetics of phase-separated condensates. We provide fluorescence-based assays to monitor the kinetics of phase separated condensates, incorporating only a small fraction of fluorescent labeling to reduce the effect of fluorescent labeling on phase separation properties.

(4) Analyzing the material properties of phase-separated condensates. We provide analytical assays in the context of the time elapsed since the condensate was first formed.

CD BioSciences is committed to developing high quality proteins, standardized procedures and eukaryotic expression systems for phase separation analysis. We provide expert protein purification solutions and guide our clients through the practicalities of in vitro protein phase separation assays, including best practice methods and pitfalls to avoid. Our services will provide a useful resource for scientists studying protein phase behavior. If you are interested in our services, please do not hesitate to contact us for more information.