1 KKETPV14 and PDZ14, which are known to induce LLPS when mixing together, are incubated with maleimide derived Alexa Fluor 488 (AF488) and sulfo Cyanine5 (Cy5) at 1:5–1:10 molar ratio, respectively.
2 Reaction mixtures are incubated at room temperature for 2 h.
3 Labeled proteins are separated from excess free fluorophores through NAP-5 column.
4 Labeling efficiency is calculated by determining concentrations of proteins and fluorophores via A280, A495 and A652.
5 Aliquot labeled proteins and store at −80 °C after flash-frozen.
6 Prepare polyethylene glycol (PEG)-passivated slides.
(A) Slides and coverslips are sonicated at 40 °C through the treatment of ethanol, KOH and ethanol.
(B) Cleaned slides and coverslips are incubated with amino-silane solution at room temperature overnight.
(C) Silanized slides and coverslips are incubated with PEGylation solution at room temperature for 2.5–3 h.
(D) Slides and coverslips are washed, dried by clean N2, vacuum sealed and stored at −20 °C.
7 Standard dye sample is used to calibrate the excitation volume of dcFCCS.
(A) The confocal microscope used to carry out dcFCCS measurements is turned on, including software and laser.
(B) Place coverslips onto the microscope objective and adjust the focal point in the solution above the coverslips.
(C) Parameters of data acquisition are set.
(D) 10 nmol/L standard dye is applied to passivated coverslips.
(E) Raw photon data is recorded for 5 min under laser excitation.
(F) Autocorrelation curve of the standard dye is used to calculate the amplitude and diffusion time. Excitation volume is then calculated from diffusion time of the standard dye.
8 Dual-labeled DNAs are prepared to calibrate correction factors of dcFCCS instrument.
(A) Purchase AF488 labeled single-stranded DNA (ssDNA) and Cy5 labeled complementary ssDNA, which are then mixed at 1:4 and 4:1 molar ratio, respectively, and annealed to obtain double-stranded DNAs (dsDNAs) as dual-labeled standard samples.
(B) As Step 7(A), turn on the microscope.
(C) As Steps 7(B)–7(C), prepare coverslips, focus lasers into solution and set parameters for data acquisition.
(D) dsDNAs containing excess AF488 labeled ssDNA or excess Cy5 labeled ssDNA are diluted to 10 nmol/L and added to passivated coverslips, respectively.
(E) Raw photon data of AF488 and Cy5 detection channels is recorded for 5 min under excitation of both 488 nm and 640 nm lasers.
(F) Autocorrelation and cross-correlation curves are calculated to determine correction factors.
9 After sample preparation and dcFCCS instrument calibration, start to acquire the data of phase separation.
(A) Prepare the microscope, coverslips and control software as shown in 7(A)–7(C).
(B) dcFCCS curves and raw photon data of 10 nmol/L AF488-KKETPV14 or 10 nmol/L Cy5-PDZ14 under 488 nm or 640 nm laser excitation, respectively, are recorded for 5 min.
(C) AF488-KKETPV14 and Cy5-PDZ14 are mixed together to induce LLPS and the mixture is added to passivated coverslips. dcFCCS curves and raw photon data of the mixture are recorded for 9 min under 488 nm and 640 nm laser excitation.
(D) After additional 41 min incubation, dcFCCS curves and raw photon data of the mixtures are recorded for another 9 min under 488 nm and 640 nm laser excitation.
10 To create a phase diagram, different concentrations of KKETPV14 and PDZ14 are mixed. Repeat the Steps 9(C)–9(D).
11 PRM14 and Cy5 labeled (SH3-KKETPV)14 are mixed to form condensates in the tube.
12 Mix client protein, AF488 labeled PDZ, into the PRM14-(SH3-KKETPV)14 phase separation system, add the whole mixture to the coverslips and record dcFCCS curves and raw photon data for 5 min under 488 nm and 640 nm laser excitation.
13 dcFCCS curves and raw photon data are used to calculate the size, growth rate, stoichiometry of condensates formed, as well as the binding affinity of client protein within condensates.
(A) Quantify the size of condensates. The diffusion times (
\begin{document}$\tau $\end{document} Dx) derived from the cross-correlation curve are used to calculate hydrodynamic radii of condensates.
(B) Quantify the growth rate of condensates. Divide the 9-min-length raw data into 1-min-length data to recalculate dcFCCS curves, from which changes of
\begin{document}$\tau $\end{document} Dx and radii over time are estimated.
(C) Quantify the molecular stoichiometry within condensates. Raw photon data is binned into 1-ms bins to generate fluorescence trajectories. By defining the threshold as three standard deviations (SDs) above the mean, only bursts exceeding the threshold are selected to calculate intensity ratio (IAF488/ICy5), which is converted into the molecular composition (NKKETPV/NPDZ) after correcting relative intensity and background.
(D) Quantify the binding affinity of the client within condensates. Amplitudes of autocorrelation and cross-correlation curves are calculated and their ratios are used to estimate the binding constant of the client within condensates.