LLPS (Temperature)
Map out the determinants of phase separation.
- Labbot
- Cuvette, internal dimensions 4x10 mm
- Micro-stir bar 1x3 mm
Many proteins and other macromolecules have the ability to self-assemble into concentrated droplets through a process known as liquid-liquid phase separation (LLPS). In recent years, LLPS has gained significant attention for its critical role in regulating various cellular functions.
The propensity for phase separation to occur is highly sensitive to solution conditions, including pH, ionic strength and temperature. Mapping out these determining factors is crucial for building an understanding of the biophysics of LLPS and gaining clues into the complex biological processes it controls.
In this application note we will look at how one can use static light scattering and the temperature titration function of the Labbot to follow the onset and the reversibility of phase separation of Ddx4 N1.
Benefits
- Light scattering gives a very sensitive measure of early phase nucleation events.
- For very turbid samples, absorbance can allow you to follow the end state.
- Automatic adjustment of temperature, finely sample the whole span.
Workflow
Setting up
Create a new procedure using the figures below as a starting point. Make any adjustments that may be needed for your experiment. Prepare your reagents.
Run the experiment
Start the experiment procedure and load your reagents. Press ‘Go’.
Get your answers
Find the plots in your Labbot data folder. View the data to find the critical aggregation temperature.
Data
Fig 1: The phase separation of Ddx4 as studied with static light scattering (blue) and absorbance (green). Both signals point to a reversal of phase separation at a NaCl concentration of around 75 mM.
Technical Note (PDF)
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Authors
Mattias Törnquist
Helping fellow scientists get the most out of their experiments with Labbot.
Alexander K. Buell
Alex is one of the leading scientists in the LLPS field. Labbot was the first instrument he acquired when setting up his new lab at DTU.
Jacob Aunstrup Larsen
M.sc. Eng. NanoBiotechnology graduate with a focus on biophysical characterization of proteins and peptides.