LLPS Starter KitLL01 Product Manual

Liquid-liquid phase separation (LLPS) is a phenomenon where two liquids with different compositions do not mix and instead separate into two distinct phases. Although this phenomenon has been well-known in physical chemistry for a long time, it has only recently been observed in living organisms, bringing an important new perspective to various biological processes.

To understand phase separation in living organisms, researchers observe phase-separated droplets in cell-free systems using purified proteins. A common method involves labeling these proteins with fluorescent markers and observing their dynamics under a microscope. Additionally, researchers study the mechanisms of droplet formation by examining how droplets react in environments with different added chemicals. With many methods available, beginners might find it challenging to decide which approach to start with.

To help beginners, we have developed an all-in-one kit based on commonly used methods for droplet formation. This kit includes all necessary reagents and containers to allow basic microscopic observations of droplets formed by phase separation in vitro using BSA (bovine serum albumin).

BSA 10 mg	× 1
BSA Dissolving Buffer (20 mmol/l HEPES pH7.4, 150 mmol/l NaCl)	× 1
Assay Buffer (60 mmol/l HEPES pH7.4, 450 mmol/l NaCl)	× 1
30% PEG8000 Solution	× 1
Slide Glass with Double-Sided Tape	× 2
Cover Glass	× 4
1.5 ml Micro Tube	× 4
0.5 ml Micro Tube	× 4

 

Store at 0–5℃

• Electronic balance
• 0.2–2 μl, 2–20 μl, 20–200 μl micropipettes
• Microscope
• Vortex Mixer
• Tweezers

• The number of phase-separated droplets may increase or decrease depending on the temperature. It is recommended to experiment at room temperature (20–25 °C).
• Please ensure that there is no dust or debris on the glass before using it.
• The edges of the cover glass can be sharp and may cause cuts. It is recommended  that gloves be worn while handling them.

Preparation of BSA Solution (800 μmol/l)

Weigh 1–2 mg of BSA into a 1.5 ml microtube. Add BSA dissolving buffer according to the formula below, then dissolve by vortexing and pipetting.

 

Example: For 2 mg of BSA, add 38 µl of BSA dissolving buffer.

 

 

 

Preparation of BSA Phase Separation Solution and Addition to the Observation Chamber

Mix the 800 µmol/l BSA stock solution, assay buffer, and PEG8000 aqueous solution in a 0.5 ml microtube. Mix by pipetting 20 times. Remove the release paper from the double-sided tape with a hole, place 2 µl of the solution in the center of the tape, and gently cover with cover glass to seal it.

• Use the same pipette tip used to measure the PEG8000 aqueous solution and keep the pipette volume set at 4 µl. Pipette gently to avoid forming bubbles. The number of pipetting actions is set to 20 to ensure thorough mixing of the solution.
• Before pipetting, if the solution appears foamy or adheres to the walls of the tube as shown below, use a tabletop centrifuge to bring the solution to the bottom of the tube, then proceed with pipetting.

 

 

 

Observation with a Microscope

It is easier to observe phase-separated droplets at the edges of the dropped solution.

 

 

Step 1: Weigh out 1–2 mg BSA.	Step 2: Add BSA Dissolving Buffer.	Step 3: Dissolve by vortexing.	Step 4: Add 2 μl BSA solution to a 0.5 μl tube.
Step 5: Add 2 µl Assay Buffer.	Step 6: Add 4 µl PEG solution.	Step 7: Pipette the solution gently 20 times, avoiding foam.	Step 8: Remove the release liner from the double-sided tape affixed to the slide glass.
Step 9: Add 2 µl solution from Step 7 to the center of the double-sided tape with a hole.
	Step 10: Gently place the cover glass over the slide glass and seal it.

 

 

 

 

 

 

Observation of phase-separated droplets using BSA
We observed the phase separation behavior under a microscope (Model: BZ-X710, KEYENCE) in 30 minutes after preparing the solution. The
phase-separated droplet formation was observed only in the condition where PEG8000 and BSA were added.