In 2012, Michael Rosen and Steven McKnight of Southwestern Medical Center discovered that molecules form droplets with weak force in a test tube, which proved for the first time that liquid-liquid phase separation (LLPS) can be repeated in vitro through simple biochemical experiments. In addition to analyzing the LLPS process in vivo through FRAP and other tests, the phase separation process can also be reconstructed in vitro under specific conditions by purifying the target protein and nucleic acid. The in vitro reconstitution experiment plays an important role in this field, and this process is also a piece of important evidence to prove the existence of LLPS mechanism.
If the driving force for in vitro assembly phase separation can be identified (for example, a protein interaction via a specific interface), then removing that driving force (for example, by changing the interface) will inhibit LLPS in vitro. Then, using these same mutants, it is possible to test whether the intracellular components are separated by the same interaction and finally form a phase separation. This kind of in vitro experiment has the obvious advantage that it can be operated with purified components and can clearly test which components are critical to the process.
Fig 1. Phase separation (Mitrea, D.M.; et al. 2018)
The advantage of in vitro reconstitution experiment is that phase separation can be monitored using a set of fully defined components. In order to exclude the possibility of contaminants affecting phase separation, the purified protein or RNA should be pure and homogeneous in size, and no polymerization should occur. In addition, phase separation is very sensitive to changes in physical and chemical conditions and requires extremely strict environmental control. Creative Proteomics can provide customers with one-stop service for phase separation in vitro reconstruction experiment, including but not limited to the following aspects.
High-quality component purification or synthesis.
Addition of kinase inhibitors, methyltransferase inhibitors, etc.
Accurately control various physical and chemical conditions such as temperature, protein, nucleic acid, or salt concentration.
We provide inverse capillary velocity, passive microrheology techniques, fluorescence correlation spectroscopy, fluorescence polarization microscope, and other mature analysis methods.
Customers can choose certain services according to project requirements, or contact us directly for consultation, and our expert team will provide you with a customized experimental program.
In order to help customers fully explore the related fields of phase separation, Creative Proteomics has recruited a large number of professionals and introduced many advanced technologies to complete phase separation in vivo analysis and in vitro reconstruction experiments. We are honored to be your competent research assistant.