Purified proteins were diluted to desired protein and salt concentrations in 50 mM HEPES pH 7,5. Dilution of salt from 1 M NaCl (storage buffer) induced phase separation. A secure seal imaging spacer (Grace Biolabs) was used between slide and coverslip to visualize protein droplets in a Leica TCS SP8 microscope. Ficoll400 (Sigma) was used in addition to induce phase separation for DL2.
For DL2:hnRNPDL-Nt colocalization experiments, DL2 was green labeled with Oregon Green and hnRNPDL-Nt was red label with Texas Red using molecular probes protein labeling kits (Invitrogen) as described in the commercial protocol. A stock at 100 μM in 150 mM NaCl was prepared for each protein and then proteins were mixed at 1:1 ratio making posterior serial dilutions from the 50 μM stock mixture.
For RNA experiments, DL1 was used at 6.25 μM final concentration to obtain droplets of 2-6 μm and ensure that the surface was not fully covered with droplets to facilitate imaging. Total RNA was obtained from HeLa cells following the TRIzol Reagent user guide (Invitrogen). RNA diluted in RNase-free water was added at the indicated concentrations and posterior addition of 5 ng/μl of RNase (Thermo Scientific) was used to induce again LLPS.
For DL2:hnRNPDL-Nt colocalization experiments, DL2 was green labeled with Oregon Green and hnRNPDL-Nt was red label with Texas Red using molecular probes protein labeling kits (Invitrogen) as described in the commercial protocol. A stock at 100 μM in 150 mM NaCl was prepared for each protein and then proteins were mixed at 1:1 ratio making posterior serial dilutions from the 50 μM stock mixture.
For RNA experiments, DL1 was used at 6.25 μM final concentration to obtain droplets of 2-6 μm and ensure that the surface was not fully covered with droplets to facilitate imaging. Total RNA was obtained from HeLa cells following the TRIzol Reagent user guide (Invitrogen). RNA diluted in RNase-free water was added at the indicated concentrations and posterior addition of 5 ng/μl of RNase (Thermo Scientific) was used to induce again LLPS.