Materials and Reagents

1.5 mL microcentrifuge tube (Axygen, catalog number: MCT-150-C)
Frosted microscope slides (Bluestar, 75 mm × 25 mm, 1.35 mm thickness)
Microscope cover glasses (Bluestar, Square 22 mm, 0.13 mm to 0.16 mm thickness)
Microcentrifuge tube holder (VWR, catalog number: 211-0207)
Purified proteins- Edc3-mCherry, Sbp1, Sbp1ΔRGG (Figure 5A) are stored at -80 °C with 10% glycerol in dialysis buffer (Roy et al., 2021). Purified proteins must be kept in aliquots at -20 °C (maximum three days as these proteins are prone to precipitation) or -80 °C (for long-term use, ≥ one week), DO NOT freeze-thaw the proteins more than two times; this might affect protein stability.
Total RNA isolated from Saccharomyces cerevisiae (Figure 5B) using the hot phenol method (store the RNA at -20 °C for short-term and -80 °C for long-term) (Garg et al., 2020).
Bovine Serum Albumin (BSA; Sigma, catalog number: A4503-100G) is stored at 4 °C.
Potassium chloride (KCl; Sigma, catalog number: P9541-1KG) is stored at room temperature.
N-(2-Hydroxyethyl) Piperazine N-(2-Ethane Sulphonic Acid) [HEPES buffer; SRL, catalog number: 16826] is stored at 4 °C.
Magnesium chloride (MgCl₂; Sigma, catalog number: M0250-1KG) is stored at room temperature.
Potassium hydroxide (KOH; Sigma, catalog number: V800320-500G) is stored at room temperature.
Diethyl pyrocarbonate (DEPC; SRL, catalog number: 46791) is stored at 4 °C.

Figure 5. Purified proteins and total RNA used in this assay. (A) Purified Edc3-mCherry, only-mCherry, Sbp1, and Sbp1ΔRGG, used for the phase separation and disassembly experiments. * Represents a degradation product of Edc3-mCherry. (B) 0.8% Formamide agarose gel electrophoresis depicting the quality of total RNA isolated from S. cerevisiae cells. Bands represent 25S rRNA, 18S rRNA, and small RNA species, respectively.
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH; Sigma, catalog number: N8129-100MG) is stored at -20 °C.
Immersion oil 1.516 NA (Cargille, catalog number: 20130)
Lint-free tissue paper (Kimwipes, Kimtech, catalog number: 34155)
500 mM KCl (For reagent preparation, see Recipes) should be stored at 4 °C.
150 mM HEPES-KOH, pH 7.4 (For buffer preparation, see Recipes), should be stored at 4 °C.
100 mM MgCl₂ (For reagent preparation, see Recipes), should be stored at 4 °C.
0.2 mM NADH (For reagent preparation, see Recipes), aliquot, and store at -80 °C. NADH is prone to degradation; therefore, it is important to aliquot 1mM NADH in nuclease-free water and store it at -80 °C.
DEPC treated H₂O/Nuclease-free H₂O (For reagent preparation, see Recipes), store at room temperature.
Phase Separation Buffer (PSB; For buffer preparation, see Recipes) must be prepared on ice. All the reaction steps must be done on ice (or 4 °C), and after reaction setup, transfer it to respective temperatures.
Absolute ethanol (EMSURE^®, Merck, catalog number: 100983)
70% ethanol (see Recipes)
DEPC treated water (see Recipes)
500 mM Potassium Chloride (KCl) (see Recipes)
150 mM HEPES-KOH buffer, pH 7.4 (see Recipes)
100 mM Magnesium chloride (MgCl₂) (see Recipes)
Phase separation buffer (PSB) (see Recipes)

Notes:

Store all the buffers at 4–8 °C. DO NOT store them at room temperature.
Wash the coverslips and glass slides with ethanol and dry them using lint-free tissue paper. Dust can hinder visualizing the phase-separated proteins.
Alternative to DEPC-treated water or solutions, commercially available RNase-free reagents can also be used for the assay.
70% ethanol is used to wash or clean the equipment. Alternatively, decontaminating agents like RNaseZap could also be used.

Equipment

Autoclavable pipettes (1–10 µL, 2–20 µL, 20–200 µL; Finpipette,Thermo Fisher Scientific, catalog number: 4701070). Clean the pipette using 70% ethanol before proceeding with the assay.
Autoclaved DNase, RNase, pyrogen-free micro tips (Axygen, catalog numbers: T-300; T-200-Y)
70% ethanol wiped scissors for cutting tip nose
30 °C incubator/heat block (Shalom, catalog number: SLM-INC-270)
Vortex Mixer (Neuation, catalog number: VM-2110)
Eppendorf^® Centrifuge 5424R (Eppendorf, catalog number: EP5404000537)
Deltavision^TM Elite Microscope (GE Healthcare) with 100× objective

Software

softWoRx 6.1.3 software for image acquisition (Applied Precision, LLC)
Fiji ImageJ Version 1.53f51 for image processing (Open source, https://imagej.net/software/fiji/downloads)
Microsoft Excel (Microsoft Corporation, USA)
GraphPad Prism 4 for data analysis (GraphPad Software Inc., https://www.graphpad.com/)

Procedure

Preparation of the phase separation experiment (15–20 min)

Thaw all the proteins and reagents on ice before proceeding with the experiment.
Keep the incubator/dry bath at 30 °C.
Pre-cool the microcentrifuge tubes by embedding them into ice before proceeding with the experiment.
Clean the workbench with 70% ethanol (alternative: use decontaminating agents like RNaseZap) before proceeding, as phase separation requires RNA, which might degrade because of RNase contamination or impurities.

Prepare LLPS buffer for a reaction mixture of 120 µL total volume, as shown in Table 1 (10 min):

Reaction I: Edc3-mCherry + PSB

Reaction II: Edc3-mCherry + 1 µg RNA + PSB

Reaction III: Edc3-mCherry + 0.2 mM NADH + PSB

Table 1. Reactions for phase separation experiment.

Reagent	Reaction I (final conc.)	Reaction I (final conc.)	Reaction III (final conc.)
500 mM KCl	150 mM	150 mM	150 mM
150 mM HEPES-KOH, pH 7.4	30 mM	30 mM	30 mM
100 mM MgCl₂	2 mM	2 mM	2 mM
1mM NADH	n/a	n/a	0.2 mM
Total RNA	n/a	1µg	n/a
Edc3-mCherry	10 µM	10 µM	10 µM
Nuclease free H₂O	Adjust volume to 120 µL	Adjust volume to 120 µL	Adjust volume to 120 µL

Phase separation reaction (Figure 3) (1 h 20 min)
1. Prepare the reaction mixtures as indicated in step A5, and keep them ready on ice.
2. Incubate the reaction mixtures at 30 °C for 1 h without disturbing the reaction tubes.
3. Take out the reaction mixtures, and gently vortex (or tap) the microcentrifuge tube to mix the phase-separated proteins.
4. Aliquot 30 µL ×3 reactions to 1.5-mL microcentrifuge tubes using a 200-µL tip with a cut (at 3–5 mm at the nozzle), and place the reactions on ice. Cutting the tip at the nozzle is critical, as it might affect the size of the phase-separated protein due to suction pressure or size restriction at its nozzle.
5. Prepare and execute the reactions from step B4 for the disassembly experiment (steps C1–C3).
6. Immediately carry forward the remaining reaction from step B3 for processing and visualization (steps D1–E6) to confirm the assembly. The processing and visualization of the phase separation reactions are discussed in steps D1–E6.
Reaction for disassembly experiment (Figure 4) (1 h 20 min)
1. Add 10 µM of purified Sbp1 or Sbp1ΔRGG or Bovine Serum Albumin (Figure 3) to the respective reactions from step B4. Mix the reaction by tapping or gentle vortexing.
2. Incubate the reaction mixtures at 30 °C for 1 h without perturbing the microcentrifuge stand or the reaction tubes.
3. Keep the reaction on ice after incubation for post-incubation processing (steps D1–D3).
Post incubation processing of the reactions (5 min)
1. Spin the reactions at 200 × g for 2 min.
2. Gently take out 20 µL of the reaction using a 200 µL tip cut at its nozzle to discard as we will not be working with the supernatant.
3. Keep the remaining reaction from step D2 on ice for visualization under the microscope (steps E1–E6).
Visualizing the phase-separated protein under the microscope (7 min–15 min)
1. Remove 5 µL of the reaction from the reaction leftover, and place it on a coverslip forming a droplet
  Note: Place the coverslip on a lint-free tissue paper to avoid capturing unwanted particles from the bench.
2. Place the glass slide on the coverslip. Fix the glass slide to the coverslip by gently putting pressure on the glass slide from the top using your thumb. Make sure the reaction gets evenly diffused between the slide and coverslip.
3. Pipette 2 µL of 1.516 NA immersion oil onto the coverslip.
4. Mount the slide on the microscope.
5. Turn the mCherry filter on (excitation: 587 λ; emission: 610 λ) and gently use the fine focusing of the microscope to focus on the assemblies. This step is time-sensitive, as the assemblies from step B6 might grow larger in size when kept at RT for a prolonged period. The imaging time should not exceed more than 7 min. It would take some time to practice focusing on the assemblies, as they are not very clearly visible in white light. We suggest performing a few mock experiments before proceeding with the main experiment. We also recommend setting up initial reactions in a staggered manner, to get enough time for visualizing each reaction.
6. The assemblies appear as depicted in Figure 3.

Data analysis

Quantitation of the phase-separated assemblies using Fiji ImageJ (Figure 6A–C).

For efficient analysis of phase-separated assembly, capture all images at the same magnification with a similar contrast setting.
Open Fiji ImageJ software, which can be downloaded from https://imagej.net/software/fiji/downloads.
In Fiji, use the menu to open an image (Figure 6D).
Select analyze, then set scale. The scale should be set by default in Fiji using the metadata; if not, enter the distance in pixels using the specifications provided by the microscope or the software. Enter the units (µm or nm). In this case, we used micrometers. Check global so that this measurement is applied to all images with the same magnification (Figure 6E).
Click on the image and duplicate the image by clicking image menu on the toolbar, then duplicate from the option.
Change the image to an 8-bit image by clicking image, then type, 8-bit (Figure 6F).
Under the image menu, select adjust, then threshold (Figure 6G).
Adjust the threshold by sliding the bars so that only the assemblies required to analyze are selected. Then provide the lower threshold and upper threshold value using the set option. Here, we used a lower threshold value of 61 and an upper threshold of 255. Keep it constant throughout the analysis (Figure 6H–I).
Under the analyze tab, select set measurements and check the measurements sufficient for data analysis (Figure 6J).
Then click on analyze and select analyze particles. Check the display results and other settings required for the data analysis. Set a minimum particle size here, so that noise is not included in the data. Then click ok (Figure 6K).
Measurements will show in a chart (Figure 6L). Copy and paste these measurements into excel to analyze the data, then create graphs using GraphPad Prism (Figure 6A–C).

Figure 6. Quantitation of phase-separated assemblies. (A) Quantitation of phase-separated assemblies depicted in Figure 3. (B) Quantitation of disassembly experiment depicted in Figure 4A. (C) Quantitation of disassembly experiment depicted in Figure 4B. (D) Menu bar of Fiji software. (E) Setting up the scale of the acquired image in Fiji. (F) Changing the bit size of the image before proceeding for analysis. (G) Adjusting the threshold for the assemblies. (H–I) Setting the threshold for the assemblies to analyze. (J) Set the kinds of measurements expected from the software. (K) Analyze particle size using the analyze particle option. (L) Copy and paste the data from the results window to the excel sheet for analysis. Data plots in Figure 4A–C represent mean ± SEM from n = 3, where ‘n’ represents the number of independent experiments. A two-tailed paired student t-test was used to calculate P-values.

Notes

Troubleshooting

Use the troubleshooting solutions in Table 2.

Table 2. Troubleshooting table

	Problem	Possible reason	Solution
1	A large aggregation of protein under the microscope	The concentration of protein might be high.	Before starting the experiment, try out different concentrations of proteins for phase separation.
		Protein might be aggregated before incubation in the PSB buffer, while being purified.	Centrifuge the purified protein at 15,000 × g for 10 min, collect the supernatant, quantify protein concentration, and use this for the phase separation experiment.
2	No phase-separated proteins were found under the microscope	Different RNA binding proteins require a particular substrate, co-factor, etc., for phase separation. Although some proteins phase-separate alone, the presence of RNA/DNA, NADH, or ATP might increase or decrease its ability of phase separation.	Try out the phase-separation of protein without any substrate with different concentrations of proteins at different time points. Try out co-factors like ATP, GTP, etc., or RNA substrates like poly(A) or poly (U), as reported earlier, to help the proteins phase separate. RNA and NADH might degrade due to some contamination in the reaction mixture. Try setting up the reaction in a clean and RNase-free environment. Check the pH and concentration of the buffer, as a change in these might lead to less or low phase-separation of proteins.
3	Aggregation of proteins during purification	Proteins containing low-complexity regions tend to aggregate during purification.	Use the L-arginine-containing lysis buffer to purify proteins (Roy et al., 2022).
4	Presence of air bubbles within the glass slide-coverslip junction	Inadequate diffusion of reaction throughout the coverslip or the glass slide was pressed too hard onto the coverslip.	Try gently placing the glass slide onto the coverslip and allowing the reaction liquid to diffuse throughout the coverslip first. Then, gently press the glass slide by rolling the thumb from one side to the other.
5	High noise signal while visualizing the phase-separated protein under the microscope.	Dust or dirt present in the glass slide or coverslip	Try cleaning the coverslip and the glass slide by first dipping it in 100% ethanol and drying it out using lint-free tissue paper. Using ordinary tissue paper could be problematic because the free-flowing lint might emit an unwanted signal.

Recipes

70% ethanol
Prepare 100 mL of 70% ethanol by adding 30 mL of autoclaved H₂O to 70 mL of absolute ethanol. Store it in a glass reagent bottle at room temperature.
DEPC treated water
Add 2 mL of DEPC (Diethyl pyrocarbonate) to 2 L of MilliQ water in the fume hood. Post addition of DEPC, store the container in a dark area overnight, and then autoclave.
500 mM Potassium Chloride (KCl)
Prepare 500 mM KCl solution by adding 3.73 g of KCl in 100 mL of autoclaved DEPC treated MilliQ water. Stir with a glass rod until all the salt dissolves. Store at 4 °C after preparation.
150 mM HEPES-KOH buffer, pH 7.4
Prepare 150 mM HEPES solution by adding 3.57 g of HEPES salt in 100 mL of autoclaved DEPC treated MilliQ water. Stir with a glass rod until all the salt dissolves. Adjust the pH of the solution to 7.4 by dropwise adding 1 M potassium hydroxide (KOH) solution using a glass pipette. Store at 4 °C after preparation.
100 mM Magnesium chloride (MgCl₂)
Prepare 100 mM MgCl₂ solution by adding 0.95 g of MgCl₂ salt to 100 mL of autoclaved DEPC treated MilliQ water. Stir with a glass rod until all the salt dissolves. Store at 4 °C after preparation.
Phase separation buffer (PSB)
Prepare the phase separation buffer according to the final volume of the reaction mixture. The final concentrations of the component buffers are as follows:
Reagent Final concentration Volume
500 mM KCl 150 mM 36 µL
150 mM HEPES-KOH, pH 7.4 30 mM 24 µL
100 mM MgCl₂ 2 mM 2.4 µL
Reaction volume n/a 120 µL
After adding PSB components, add the required amount of purified protein or RNA into the reactions separately (Table 1), and make up the volume to 120 µL using autoclaved DEPC treated MilliQ water.

Acknowledgments

We acknowledge support from the DBT/Wellcome Trust India Alliance Fellowship/Grant [IA/I/12/2/500625] and DBT India grant [BT/PR40106/BRB/10/1918/2020]. We also thank the DBT-IISc partnership program and DST-FIST program for infrastructure support. DBT-JRF for fellowship to RR. This protocol was previously used to understand the mechanism of disassembly by Sbp1 in the article titled “Low complexity RGG motif sequence is required for Processing body (P-body) disassembly.” We thank Amrendra Kumar for helping us troubleshoot the purification of Edc3-mCherry protein. We thank Rajyaguru lab members for their constant input, support, and encouragement.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics

The authors declare no conflict with respect to ethical grounds.

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