In vitro phase separation assay

In vitro phase separation

In Zhang et al., 2022, we performed in vitro phase separation with various GFP-tagged recombinant proteins. Here, we take GFP and GFP-HRLP as examples for this detailed protocol of “in vitro phase separation”. 

1. Clone the coding sequences of GFP and GFP fused with HRLP into the pGEX-6p-2 vector (Pharmacia) to generate GST-GFP and GST-GFP-HRLP constructs, respectively.

2. Transform the constructs of GST-GFP and GST-GFP-HRLP into Escherichia coli Rosetta (DE3) competent cells.

3. Inoculate one single colony into 2 ml of LB liquid medium supplemented with appropriate antibiotics and incubate at 37°C overnight with vigorous shaking.

4. Transfer the overnight cell culture to 200 ml of LB liquid medium supplemented with antibiotics and grow at 37°C until the OD₆₀₀ value reach 0.6 - 1.0. 

5. Add 0.2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) to the cultured cells to induce proteins of interest at 16°C for overnight. After IPTG induction, the colour of the cells usually turns into green, which is an indicator of successful induction of GST-GFP and GST-GFP-HRLP fusion proteins.

6. Test the solubility of GST-GFP and GST-GFP-HRLP fusion proteins by running a protein gel followed by Coomassie blue staining. 

7. After confirming the solubility of proteins, harvest the induced cells containing GST-GFP and GST-GFP-HRLP by centrifugation at 3,500 rpm for 5 min at 4°C. 

8. Resuspend the cells in 10 ml of ice-cold lysis buffer, followed by sonication until the cell suspension becomes clear. 

9. Centrifuge the suspension at 10,000 rpm for 20 min at 4°C.

10. Transfer the supernatant containing induced proteins into new 15 ml Falcon tubes.

11. Add 150 μl of Glutathione Sepharose beads (Amersham Bioscience) to each supernatant, followed by an incubation for 2 hr at 4°C with gentle rotation. 

12. Centrifuge for 1 min at 1,000 rpm at 4°C to pellet the beads. 

13. Discard the supernatants and wash beads with 5 ml of ice-cold lysis buffer for 5 min.

14. Repeat the washing step twice.

15. Test the protein quality and size by running 2 μl of washed beads on a protein gel followed by Coomassie blue staining.

16. Add 2 μl of PreScission Protease (GE Healthcare) and 200 μl cleavage buffer to the washed beads.

17. Incubate the beads on a nutating mixer at 4°C overnight.

18. Centrifuge the beads for 1 min at 2,500 rpm, 4°C.

19. Transfer the supernatants to new 1.5 ml tubes to store GFP and GFP-HRLP proteins. The proteins can be stored in aliquots at -80°C for several months.

20. Measure the protein concentration with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad).

21. Run 2 μl of GFP and GFP-HRLP proteins on a protein gel followed by Coomassie blue staining to check the protein quality and size.

22. Dilute generated GFP and GFP-HRLP proteins at the final concentration of 10 μM with the cleavage buffer on ice. 

23. Add PEG-8000 (Sigma-Aldrich) at the final concentration of 10% to the diluted proteins in a PCR tube on ice. The concentration of protein or PEG-8000 could be adjusted.

24. Transfer the mixture to a Hole Microscope Slide immediately to avoid protein degradation or fluorescence decay.

25. Carefully cover the slide with a cover glass.

26. Observe the fluorescence with 40X microscope objective under an FV3000 Olympus confocal microscope. GFP protein exhibits uniform signals under the microscope, whereas GFP-HRLP forms droplets. 

Solutions:

Coomassie blue staining buffer:

20% methanol, 10% acetic acid and 0.1% Coomassie Brilliant Blue R-250

Lysis buffer:

10 mM Tris-HCl pH 8.0, 15 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5 mM DTT, 1x Complete EDTA-free Protease Inhibitor Cocktail (Roche)

Cleavage buffer:

50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Triton X-100