# Droplet or clump? Phase separation of the prion protein > Many proteins undergo liquid–liquid phase separation (LLPS), forming dense droplets without a membrane. Source: https://khushisingh.in/research/prion-phase-separation/ Author: Khushi Singh (khushisingh.in). Licence: CC BY 4.0 for text and original illustrations, unless a credit says otherwise. Evidence labels used on this site: established, strong evidence, emerging, hypothesis, debated. Where a page has Simple, Curious and Expert versions, all three follow. --- [Research](https://khushisingh.in/research/) · Completed · May–July 2024 IISER Pune · Prof. Jayant B. Udgaonkar Proteins from: [*Mus musculus*](https://khushisingh.in/research/model-system/#mus-musculus) The question Under which conditions of salt, pH and concentration does the mouse prion protein form liquid-like droplets or aggregates? Many proteins undergo liquid–liquid phase separation (LLPS), forming dense droplets without a membrane. For the prion protein, the conditions that favour droplets versus aggregates matter because misfolded prion protein causes neurodegenerative prion diseases. I varied salt, pH and protein concentration and followed phase behaviour with UV–visible spectrophotometry. [The basics: droplets inside cells](https://khushisingh.in/explore/06-droplets-inside-cells/) ## Why ask this? Proteins are not always dissolved evenly. Many can **phase-separate** into dense, liquid-like droplets, much as oil separates from vinegar. (evidence: established) For the **prion protein** this matters: when it misfolds and aggregates, it can template its bad shape onto other copies, causing rare but devastating brain diseases. (evidence: established) Whether the protein forms reversible droplets or harmful clumps depends on its surroundings. ## From droplets to solids Phase separation starts when a protein’s concentration and its surroundings cross a **phase boundary**: the solution splits into a dense, protein-rich phase and a dilute one (Banani et al. 2017; Alberti et al. 2019). Liquid droplets are round, fuse and exchange molecules with their surroundings; with time, some harden into gels or solids (Patel et al. 2015). (evidence: established) The prion protein has a long **disordered N-terminal region** and a folded C-terminal domain. It forms a liquid phase in vitro (Kostylev et al. 2018). Its droplets can age into a β-sheet-rich, proteinase-resistant gel, which needed the N-terminal region (amino acids 23–89) and kosmotropic (water-structuring) salts (Tange et al. 2021). (evidence: emerging) Phase separation also changes the shape of the N-terminal region itself (Pal & Udgaonkar 2026). (evidence: emerging) *[Here the web page has an interactive activity; open the source URL to use it.]* Droplets scatter light, so **turbidity** is a simple readout of phase separation. That is why I followed it by UV–visible spectrophotometry. ## What I did - I changed **salt**, **pH** and **protein concentration** systematically. - I followed droplet formation and aggregation by **UV–visible spectrophotometry** (turbidity). - I read into protein misfolding and its links to neurodegeneration. ## What I took away Physics and chemistry decide biology’s states. Small changes in conditions can tip a protein from a healthy droplet into a harmful clump. The basics are in [droplets inside cells](https://khushisingh.in/explore/06-droplets-inside-cells/). ## Why it matters Understanding how condensates *age* or *solidify* is an active frontier in neurodegeneration research. (evidence: emerging) ## Methods used - Protein biochemistry - UV–visible spectrophotometry (turbidity) - Varying solution conditions (salt, pH, concentration) [What these methods are →](https://khushisingh.in/research/toolbox/) ## References 1. Pal S, Udgaonkar JB (2026) Phase separation leads to significant conformational change in the N-terminal region of the prion protein. Protein Science 35:e70742. [doi:10.1002/pro.70742](https://doi.org/10.1002/pro.70742) 2. Banani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular condensates: organizers of cellular biochemistry. Nature Reviews Molecular Cell Biology 18:285–298. [doi:10.1038/nrm.2017.7](https://doi.org/10.1038/nrm.2017.7) 3. Alberti S, Gladfelter A, Mittag T (2019) Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176:419–434. [doi:10.1016/j.cell.2018.12.035](https://doi.org/10.1016/j.cell.2018.12.035) 4. Patel A, Lee HO, Jawerth L, et al., Alberti S (2015) A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162:1066–1077. [doi:10.1016/j.cell.2015.07.047](https://doi.org/10.1016/j.cell.2015.07.047) 5. Kostylev MA, Tuttle MD, Lee S, et al., Strittmatter SM (2018) Liquid and hydrogel phases of PrPC linked to conformation shifts and triggered by Alzheimer’s amyloid-β oligomers. Molecular Cell 72:426–443. [doi:10.1016/j.molcel.2018.10.009](https://doi.org/10.1016/j.molcel.2018.10.009) 6. Tange H, Ishibashi D, Nakagaki T, et al., Nishida N (2021) Liquid–liquid phase separation of full-length prion protein initiates conformational conversion in vitro. Journal of Biological Chemistry 296:100367. [doi:10.1016/j.jbc.2021.100367](https://doi.org/10.1016/j.jbc.2021.100367)